Autophagy-related 1: Biological Overview | References
Gene name - Autophagy-related 1
Cytological map position - 69E2-69E4
Function - signaling
Keywords - autophagy-specific protein kinase
Symbol - Atg1
FlyBase ID: FBgn0260945
Genetic map position - 3L: 12,777,529..12,798,214 [-]
Classification - Serine/Threonine protein kinase
Cellular location - cytoplasmic
|Recent literature||Kuhn, H., Sopko, R., Coughlin, M., Perrimon, N. and Mitchison, T. (2015). The Atg1-Tor pathway regulates yolk catabolism in Drosophila embryos. Development [Epub ahead of print]. PubMed ID: 26395483
Yolk provides an important source of nutrients during the early development of oviparous organisms. It is composed mainly of vitellogenin proteins packed into membrane-bound compartments called yolk platelets. Catabolism of yolk is initiated by acidification of the yolk platelet, leading to the activation of Cathepsin-like proteinases, but it is unknown how this process is triggered. Yolk catabolism initiates at cellularization in Drosophila melanogaster embryos. Using maternal shRNA technology this, study found that yolk catabolism depends on the Tor pathway and on the autophagy-initiating kinase Atg1. While Atg1 was required for a burst of spatially-regulated autophagy during late cellularization, autophagy was not required for initiating yolk catabolism. It is proposed that the conserved Tor metabolic sensing pathway regulates yolk catabolism, similar to Tor-dependent metabolic regulation on the lysosome.
|Li, M., Lindblad, J. L., Perez, E., Bergmann, A. and Fan, Y. (2016). Autophagy-independent function of Atg1 for apoptosis-induced compensatory proliferation. BMC Biol 14: 70. PubMed ID: 27542914
ATG1 belongs to the Uncoordinated-51-like kinase protein family. Members of this family are best characterized for roles in macroautophagy and neuronal development. Apoptosis-induced proliferation (AiP) is a caspase-directed and JNK-dependent process which is involved in tissue repair and regeneration after massive stress-induced apoptotic cell loss. Under certain conditions, AiP can cause tissue overgrowth with implications for cancer. This study shows that Atg1 in Drosophila (dAtg1) has a previously unrecognized function for both regenerative and overgrowth-promoting AiP in eye and wing imaginal discs. dAtg1 acts genetically downstream of and is transcriptionally induced by JNK activity, and it is required for JNK-dependent production of mitogens such as Wingless for AiP. Interestingly, this function of dAtg1 in AiP is independent of its roles in autophagy and in neuronal development. It is concluded that, in addition to a role of dAtg1 in autophagy and neuronal development, a third function of dAtg1 for AiP is reported.
|Wen, J. K., Wang, Y. T., Chan, C. C., Hsieh, C. W., Liao, H. M., Hung, C. C. and Chen, G. C. (2017). Atg9 antagonizes TOR signaling to regulate intestinal cell growth and epithelial homeostasis in Drosophila. Elife 6. PubMed ID: 29144896
Autophagy is essential for maintaining cellular homeostasis and survival under various stress conditions. Autophagy-related gene 9 (Atg9) encodes a multipass transmembrane protein thought to act as a membrane carrier for forming autophagosomes. However, the molecular regulation and physiological importance of Atg9 in animal development remain largely unclear. This study generated Atg9 null mutant flies and found that loss of Atg9 led to shortened lifespan, locomotor defects, and increased susceptibility to stress. Atg9 loss also resulted in aberrant adult midgut morphology with dramatically enlarged enterocytes. Interestingly, inhibiting the TOR signaling pathway rescued the midgut defects of the Atg9 mutants. In addition, Atg9 interacted with PALS1-associated tight junction protein (Patj), which associates with TSC2 to regulate TOR activity. Depletion of Atg9 caused a marked decrease in TSC2 levels. These findings revealed an antagonistic relationship between Atg9 and TOR signaling in the regulation of cell growth and tissue homeostasis.
|Clark, S. G., Graybeal, L. L., Bhattacharjee, S., Thomas, C., Bhattacharya, S. and Cox, D. N. (2018). Basal autophagy is required for promoting dendritic terminal branching in Drosophila sensory neurons. PLoS One 13(11): e0206743. PubMed ID: 30395636
Relatively little is known regarding the developmental role of basal autophagy in directing aspects of dendritic arborization. This study demonstrates that autophagy-related (Atg) genes are positively regulated by the homeodomain transcription factor Cut, and that basal autophagy functions as a downstream effector pathway for Cut-mediated dendritic terminal branching in Drosophila multidendritic (md) sensory neurons. Further, loss of function analyses implicate Atg genes in promoting cell type-specific dendritic arborization and terminal branching, while gain of function studies suggest that excessive autophagy leads to dramatic reductions in dendritic complexity. The Atg1 initiator kinase interacts with the dual leucine zipper kinase (DLK) pathway by negatively regulating the E3 ubiquitin ligase Highwire and positively regulating the MAPKKK Wallenda. Finally, autophagic induction partially rescues dendritic atrophy defects observed in a model of polyglutamine toxicity. Collectively, these studies implicate transcriptional control of basal autophagy in directing dendritic terminal branching and demonstrate the importance of homeostatic control of autophagic levels for dendritic arbor complexity under native or cellular stress conditions.
|Nandy, A., Lin, L., Velentzas, P. D., Wu, L. P., Baehrecke, E. H. and Silverman, N. (2018). The NF-kappaB factor Relish regulates Atg1 expression and controls autophagy. Cell Rep 25(8): 2110-2120.e2113. PubMed ID: 30463009
Macroautophagy and cell death both contribute to innate immunity, but little is known about how these processes integrate. Drosophila larval salivary glands require autophagy for developmentally programmed cell death, and innate immune signaling factors increase in these dying cells. This study shows that the nuclear factor kappaB (NF-kappaB) factor Relish, a component of the immune deficiency (Imd) pathway, is required for salivary gland degradation. Surprisingly, of the classic Imd pathway components, only Relish and the PGRP receptors were involved in salivary gland degradation. Significantly, Relish controls salivary gland degradation by regulating autophagy but not caspases. In addition, expression of either Relish or PGRP-LC causes premature autophagy induction and subsequent gland degradation. Relish controls autophagy by regulating the expression of Atg1, a core component and activator of the autophagy pathway. Together these findings demonstrate that a NF-kappaB pathway regulates autophagy during developmentally programmed cell death.
|Gunaseelan, S., Wang, Z., Tong, V. K. J., Ming, S. W. S., Razar, R., Srimasorn, S., Ong, W. Y., Lim, K. L. and Chua, J. J. E. (2021). Loss of FEZ1, a gene deleted in Jacobsen syndrome, causes locomotion defects and early mortality by impairing motor neuron development. Hum Mol Genet. PubMed ID: 33395696
FEZ1-mediated axonal transport plays important roles in central nervous system development but its involvement in the peripheral nervous system is not well-characterised. FEZ1 is deleted in Jacobsen syndrome (JS), an 11q terminal deletion developmental disorder. JS patients display impaired psychomotor skills, including gross and fine motor delay, suggesting that FEZ1 deletion may be responsible for these phenotypes, given its association with the development of motor-related circuits. Supporting this hypothesis, the data shows that FEZ1 is selectively expressed in the rat brain and spinal cord. Its levels progressively increase over the developmental course of human motor neurons derived from embryonic stem cells. Deletion of FEZ1 strongly impaired axon and dendrite development, and significantly delayed the transport of synaptic proteins into developing neurites. Concurring with these observations, Drosophila unc-76 mutants showed severe locomotion impairments, accompanied by a strong reduction of synaptic boutons at neuromuscular junctions. These abnormalities were ameliorated by pharmacological activation of UNC-51/ATG1, a FEZ1-activating kinase, with rapamycin and metformin. Collectively, the results highlight a role for FEZ1 in motor neuron development and implicate its deletion as an underlying cause of motor impairments in JS patients.
|Bjedov, I., Cocheme, H. M., Foley, A., Wieser, D., Woodling, N. S., Castillo-Quan, J. I., Norvaisas, P., Lujan, C., Regan, J. C., Toivonen, J. M., Murphy, M. P., Thornton, J., Kinghorn, K. J., Neufeld, T. P., Cabreiro, F. and Partridge, L. (2020). Fine-tuning autophagy maximises lifespan and is associated with changes in mitochondrial gene expression in Drosophila. PLoS Genet 16(11): e1009083. PubMed ID: 33253201
Increased cellular degradation by autophagy is a feature of many interventions that delay ageing. This paper reports that increased autophagy is necessary for reduced insulin-like signalling (IIS) to extend lifespan in Drosophila and is sufficient on its own to increase lifespan. It was first established that the well-characterised lifespan extension associated with deletion of the insulin receptor substrate chico was completely abrogated by downregulation of the essential autophagy gene Atg5. Next autophagy was directly induced by over-expressing the major autophagy kinase Atg1; a mild increase in autophagy extended lifespan. Interestingly, strong Atg1 up-regulation was detrimental to lifespan. Transcriptomic and metabolomic approaches identified specific signatures mediated by varying levels of autophagy in flies. Transcriptional upregulation of mitochondrial-related genes was the signature most specifically associated with mild Atg1 upregulation and extended lifespan, whereas short-lived flies, possessing strong Atg1 overexpression, showed reduced mitochondrial metabolism and up-regulated immune system pathways. Increased proteasomal activity and reduced triacylglycerol levels were features shared by both moderate and high Atg1 overexpression conditions. These contrasting effects of autophagy on ageing and differential metabolic profiles highlight the importance of fine-tuning autophagy levels to achieve optimal healthspan and disease prevention.
|Deehan, M., Lin, W., Blum, B., Emili, A. and Frydman, H. (2021). Intracellular Density of Wolbachia Is Mediated by Host Autophagy and the Bacterial Cytoplasmic Incompatibility Gene cifB in a Cell Type-Dependent Manner in Drosophila melanogaster. mBio 12(1). PubMed ID: 33436431
Autophagy is an intracellular degradation pathway involved in innate immunity. Pathogenic bacteria have evolved several mechanisms to escape degradation or exploit autophagy to acquire host nutrients. In the case of endosymbionts, which often have commensal or mutualistic interactions with the host, autophagy is not well characterized. This study utilized tissue-specific autophagy mutants to determine if Wolbachia, a vertically transmitted obligate endosymbiont of Drosophila melanogaster, is regulated by autophagy in somatic and germ line cell types. This analysis revealed core autophagy proteins Atg1 and Atg8 and a selective autophagy-specific protein Ref(2)p negatively regulate Wolbachia in the hub, a male gonad somatic cell type. Furthermore, it was determined that the Wolbachia effector protein, CifB, modulates autophagy-Wolbachia interactions, identifying a new host-related pathway which these bacterial proteins interact with. In the female germ line, the cell type necessary for inheritance of Wolbachia through vertical transmission, it was discovered that bulk autophagy mediated by Atg1 and Atg8 positively regulates Wolbachia density, whereas Ref(2)p had no effect. Global metabolomics of fly ovaries deficient in germ line autophagy revealed reduced lipid and carbon metabolism, implicating metabolites from these pathways as positive regulators of Wolbachia. This work provides further understanding of how autophagy affects bacteria in a cell type-dependent manner.
|Ham, S. J., Lee, D., Xu, W. J., Cho, E., Choi, S., Min, S., Park, S. and Chung, J. (2021). Loss of UCHL1 rescues the defects related to Parkinson's disease by suppressing glycolysis. Sci Adv 7(28). PubMed ID: 34244144
The role of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1; also called PARK5) in the pathogenesis of Parkinson's disease (PD) has been controversial. This study finds that the loss of UCHL1 destabilizes pyruvate kinase (PKM) and mitigates the PD-related phenotypes induced by PTEN-induced kinase 1 (PINK1) or Parkin loss-of-function mutations in Drosophila and mammalian cells. In UCHL1 knockout cells, cellular pyruvate production and ATP levels are diminished, and the activity of AMP-activated protein kinase (AMPK) is highly induced. Consequently, the activated AMPK promotes the mitophagy mediated by Unc-51-like kinase 1 (ULK1) and FUN14 domain-containing 1 (FUNDC1), which underlies the effects of UCHL1 deficiency in rescuing PD-related defects. Furthermore, this study identified tripartite motif-containing 63 (TRIM63) as a previously unknown E3 ligase of PKM and demonstrate its antagonistic interaction with UCHL1 to regulate PD-related pathologies. These results suggest that UCHL1 is an integrative factor for connecting glycolysis and PD pathology.
To survive starvation and other forms of stress, eukaryotic cells undergo a lysosomal process of cytoplasmic degradation known as autophagy. Autophagy has been implicated in a number of cellular and developmental processes, including cell growth control and programmed cell death. However, direct evidence of a causal role for autophagy in these processes is lacking, due in part to the pleiotropic effects of signaling molecules such as TOR that regulate autophagy. This study circumvents this difficulty by directly manipulating autophagy rates in Drosophila through the autophagy-specific protein kinase Atg1 (Atg signifies an autophagy-related gene). Overexpression of Atg1 is sufficient to induce high levels of autophagy, the first such demonstration among wild type Atg proteins. In contrast to findings in yeast, induction of autophagy by Atg1 is dependent on its kinase activity. Cells with high levels of Atg1-induced autophagy are rapidly eliminated, demonstrating that autophagy is capable of inducing cell death. However, this cell death is caspase dependent and displays DNA fragmentation, suggesting that autophagy represents an alternative induction of apoptosis, rather than a distinct form of cell death. In addition, this study demonstrates that Atg1-induced autophagy strongly inhibits cell growth, and that Atg1 mutant cells have a relative growth advantage under conditions of reduced TOR signaling. Finally, this study shows that Atg1 expression results in negative feedback on the activity of TOR itself. These results reveal a central role for Atg1 in mounting a coordinated autophagic response, and demonstrate that autophagy has the capacity to induce cell death. Furthermore, this work identifies autophagy as a critical mechanism by which inhibition of TOR signaling leads to reduced cell growth (Scott, 2007).
Under starvation conditions, eukaryotic cells recover nutrients via autophagy, a lysosome-mediated process of bulk cytoplasmic degradation. Through autophagy, long-lived proteins, organelles, and other components of the cytoplasm are non-selectively engulfed within specialized double-membraned vesicles known as autophagosomes. Subsequent fusion of the outer autophagosomal membrane with the lysosome results in a structure known as the autolysosome, in which the inner membrane and its cytoplasmic cargo are degraded. Breakdown products released from the autolysosome supply the cell with an internal source of nutrients that can support essential metabolic processes during starvation. Approximately 20 ATG (autophagy-related) genes specifically required for autophagy have been discovered in Saccharomyces cerevisiae (Klionsky, 2003), and many of these genes have functional homologs in metazoans (Levine, 2004; Scott, 2007 and references therein).
In addition to survival of starvation, autophagy has been implicated in many aspects of health and development, including aging, programmed cell death, pathogenic infection, stress responses, neurodegenerative and muscle disorders, cellular remodeling, cancer, and cell growth (Levine, 2004; Shintani, 2004). As in many of these processes, evidence for a role of autophagy in cell growth control is largely correlative. Autophagy is promoted by several tumor suppressor genes including PTEN, TSC1, TSC2, and beclin 1, and is inhibited by growth-promoting pathways such as type I PI3K and target of rapamycin (TOR) signaling. A variety of conditions that stimulate cell growth, such as growth factor addition, partial hepatectomy, and refeeding after starvation, also inhibit autophagy, while growth suppressive signals, such as contact inhibition and substrate detachment, induce autophagy (Newfeld, 2004). Together, these correlative findings are consistent with a model in which the catabolic effects of autophagy act as a brake on cell growth during development. However, this issue is complicated by the pleiotropic nature of the signaling pathways that regulate autophagy. For example, in addition to inhibiting autophagy, the TOR pathway controls cell metabolism and biosynthesis by promoting ribosome biogenesis, protein synthesis and nutrient uptake. The relevant contribution of each of these downstream effector pathways to net cell growth is poorly understood (Scott, 2007).
Autophagy is also known to be involved in programmed cell death, and distinguishes Type II (autophagic) from Type I (apoptotic) cell death. Autophagic cell death is characterized by an abundance of autophagosomes and autolysosomes in the dying cell and differs from apoptotic cell death in that dying cells are degraded by their own lysosomal enzymes, rather than by phagocytosis. It has been difficult, however, to establish whether autophagy plays a causal role in Type II cell death or represents a failed attempt at cell survival in cells undergoing programmed cell death. Death signals often induce features of both apoptosis and autophagy, and mutations that disrupt autophagy have been shown to suppress cell death in some cases (Shimizu, 2004; Veneault-Fourrey, 2006, Yu, 2006), and hasten it in others (see Lum, 2005). Thus, as in the case for cell growth, much of the support for a direct role for autophagy in cell death rests on correlative evidence (Scott, 2007).
One approach to addressing the potential role of autophagy in functions such as cell death and growth would be to induce autophagy directly, independent of the signaling pathways that normally control it. In yeast, multiple signaling pathways, including TOR, AMPK and Ras/PKA, converge on the Ser-Thr kinase Atg1 to regulate autophagy (Kamada, 2000; Wang, 2001; Budovskaya, 2005). In addition, Atg1 interacts with multiple components of the autophagic machinery, through direct association, phosphorylation, and/or through effects on intracellular localization (Kamada, 2000; Ptacek, 2005; Reggiori, 2004). Thus, Atg1 may represent a nodal point for controlling multiple steps in the autophagic process in response to various inductive cues (Scott, 2007).
Interestingly, the role of Atg1 kinase activity in yeast is the subject of some debate, and studies from different groups using ATP analog-sensitive and kinase-defective Atg1 mutants have reached conflicting conclusions. One study reported that Atg1 kinase activity is required for the autophagy-related biosynthetic cytoplasm to vacuole targeting (CVT) pathway but not for autophagy, and concluded that Atg1 plays a structural role in autophagy (Abeliovich, 2003). However, other studies using similar reagents found a requirement for Atg1 kinase activity in both CVT and autophagy (Kamada, 2000; Kabeya, 2005; Scott, 2007 and references therein).
It has been shown that the Drosophila homolog of Atg1 is required for autophagy in the larval fat body, an organ analogous to the vertebrate liver with roles in nutrient storage and mobilization. This study investigated the effects of Drosophila Atg1 loss of function and overexpression on autophagy induction, cell growth control, and cell death. The findings indicate that Atg1 expression is sufficient to effect a full autophagic response, resulting in a marked inhibition of cell growth and a rapid induction of apoptotic cell death (Scott, 2007).
Delivery of cytoplasmic components to the lysosome through autophagy involves multiple distinct steps including nucleation, expansion and closure of the autophagosome, and its subsequent fusion with the lysosome. These membrane trafficking events require the recruitment and subsequent retrieval of a large number of autophagy-specific Atg proteins, as well as general factors involved in vesicle trafficking. Given this complexity, the finding that overexpression of a single Atg protein is sufficient to accomplish all essential steps in this process is striking. As autophagy occurs constitutively at a basal rate in most eukaryotic cells, acceleration of a single rate-limiting step by overexpression of Atg1 may be sufficient to increase the overall rate of autophagy. Alternatively, the interaction of Atg1 with multiple Atg proteins suggests that Atg1 may function at multiple steps in the autophagic process. Identification of the relevant in vivo substrates of Atg1 will help to clarify this issue. A recent yeast proteomic microarray study (Ptacek, 2005) identified a number of in vitro substrates of Atg1, including Atg8 and Atg18, which function in autophagosomal expansion and retrieval of components of the autophagic machinery, respectively, as well as general factors involved in vesicle transport and vacuolar function (Scott, 2007).
The role of the kinase activity of yeast Atg1 in regulating autophagy is the matter of some debate, since different groups have drawn conflicting conclusions. A consensus view, however, may be that autophagy and the CVT pathway require different levels of Atg1 kinase activity, or that Atg1 has different substrates under differing nutrient conditions. In higher eukaryotes, the role of Atg1 kinase activity may differ from that in yeast, as the CVT pathway has not been observed in metazoan cells. Consistent with the findings of Ohsumi and colleagues (Kamada, 2000; Kabeya, 2005), however, the current results indicate that the kinase domain of Drosophila Atg1 is required for autophagy, since the kinase-inactive Atg1K38Q mutant failed to restore starvation-induced autophagy to Atg1 mutants, and was unable to induce autophagy when overexpressed. However, the partial reduction in size and TOR activity observed upon expression of Atg1K38Q, as well as its partial rescue of Atg1 mutants, indicate that Atg1 may have other, kinase-independent functions beyond regulating autophagy (Scott, 2007).
The mechanisms by which upstream signaling pathways regulate Atg1 are also likely to differ between yeast and multicellular organisms; essential components of the TOR-regulated Atg1 complex such as Atg13 and Atg17 are not readily identifiable in animal genomes. Nonetheless, the current results are consistent with a similar negative regulation of Atg1 by TOR, since activation of TOR signaling by Rheb overexpression or Tsc2 mutation suppresses the ability of Atg1 to induce autophagy. Increased Atg1 activity in response to loss of TOR signaling is likely to be critical for cell homeostasis and survival, since animals doubly mutant for Atg1 and Tor show a synthetic embryonic lethal phenotype (Scott, 2004). In addition, the results demonstrate an unexpected mode of signaling from Atg1 to TOR, in which increased Atg1 levels lead to downregulation of TOR kinase activity. It is unclear whether this reflects a direct effect of Atg1 on the activity of TOR or an upstream regulator, or an indirect consequence of the high level of autophagy in these cells, perhaps through increased turnover of TOR signaling components. The finding that a pool of TOR protein resides on intracellular vesicles and that TOR signaling is reduced in endocytic mutants suggests that TOR activity may respond to rates of vesicular trafficking, such as autophagy. Regardless of mechanism, these results suggest the existence of a self-reinforcing feedback loop, whereby increased Atg1 levels lead to downregulation of TOR activity, resulting in further activation of Atg1. In contrast, induction of autophagy in response to loss of TOR signaling is dampened by the resultant inactivation of S6K, which is required for normal autophagy (Scott, 2004). Thus TOR-mediated regulation of autophagy involves both positive and negative feedback (Scott, 2007).
Autophagy is a catabolic process that inversely correlates with cell growth, suggesting that increased levels of autophagy observed in growth-restricted cells may contribute to their reduced growth rate. The results presented here provide genetic support for this model. The data indicate that clones of autophagy-defective cells have a growth advantage over wild type cells under physiological conditions that normally induce autophagy, including starvation and rapamycin treatment. These results are confirmed by the marked size reduction of cells overexpressing Atg1, further supporting the role of autophagy as a negative effector of growth in TOR signaling. Thus, autophagy is partly responsible for the growth restriction resulting from physiological inhibition of TOR signaling, and is capable of inhibiting growth independent of TOR signaling (Scott, 2007).
In contrast to the growth advantage observed in autophagy defective cells, previous studies using yeast or cultured mammalian cells reported that inhibition of autophagy results in rapid cell death in response to starvation. This difference is likely due to the mosaic nature of the current experiments, in which clones of Atg1 mutant cells are surrounded by autophagy-competent wild type cells, and may in effect parasitize nutrients liberated through the autophagic activity of their neighbors. This is likely to be particularly evident in fat body cells, which are specialized for nutrient storage and mobilization. It is noted that this genetic mosaicism is similar to the situation facing tumor cells within wild type tissues. Thus, the increased growth capacity resulting from disruption of autophagy may contribute to the tumorigenicity of cells mutant for tumor suppressors such as PTEN, TSC1 & 2, beclin 1/Atg6 and possibly LC3/Atg8 and Atg7 (Scott, 2007 and references therein).
Whereas autophagy had an inhibitory effect on cell growth under physiological conditions, this was not the case in cells with severely disrupted TOR signaling, such as in cells with null mutations in Pdk1 Tor, despite the strong correlation of reduced cell growth and increased autophagy in these cells. Disruption of autophagy had no effect on the growth of Pdk1 null cells, and actually led to a further decrease in cell size of Tor mutants. In the complete absence of TOR signaling, nutrient uptake is severely curtailed, and under these extreme conditions the metabolic benefits of nutrients liberated by autophagy may outweigh its potential growth-inhibitory catabolic effect. It is concluded that autophagy has context-dependent effects on cell growth, providing for a rudimentary level of metabolism and growth under conditions of severe nutrient deprivation, acting as a net inhibitor of growth under conditions of reduced TOR signaling, and strongly inhibiting cell growth when induced to high levels. These findings add autophagy to the growing list of effector pathways and cellular processes through which TOR signaling controls cell growth, including translation, transcription, nutrient import and endocytosis (Scott, 2007).
Previous studies in a number of experimental systems indicate that the role of autophagy in cell death is also likely to be context-dependent. For example, autophagy has been found to protect against cell death in cases of growth factor withdrawal, starvation, and neurodegeneration, but to be required for some cases of autophagic cell death. Thus, observations of autophagic structures in dying cells are equally consistent with a causal, neutral or even inhibitory role of autophagy in cell death. The ability to induce autophagy through Atg1 overexpression has enabled a direct test of the potential role of autophagy in promoting cell death. The results indicate that induction of autophagy is sufficient to induce cell death. It was found that death resulting from Atg1-induced autophagy is suppressed by caspase inhibition and is associated with caspase activation, DNA fragmentation, and cytoskeletal disruption, suggesting that high levels of autophagy result in apoptotic cell death. The connection between apoptosis and autophagy is further supported by the recent demonstration that overexpression of the anti-apoptotic protein Bcl-2 can inhibit autophagy by interacting with the Atg6 homolog Beclin 1 (Pattingre, 2005). Mutant versions of Beclin 1 that are unable to bind Bcl-2 stimulate autophagy and promote cell death, similar to the effects of Atg1 (Scott, 2007).
By what mechanisms might autophagy lead to cell death? The observation that starvation-induced autophagy is reversible and does not normally result in cell elimination suggests that starvation conditions may be protective against autophagy-induced death. Induction of autophagy is tolerated or even beneficial in cells with reduced biosynthetic activity, but may be detrimental in cells whose resources are devoted to continued growth. Self-limiting mechanisms also serve to prevent starvation-induced autophagy from proceeding at continuously high levels. During autophagic cell death, alternate activation of the autophagic machinery may circumvent these feedback mechanisms, resulting in high levels of sustained autophagy that are destructive to the cell. In addition, the level of Atg1 gene expression may be critical, since it is not upregulated under starvation conditions (Kirisako, 1999), but its levels peak at the beginning of the Drosophila pupal stage, when autophagic cell death is induced. Autophagic cell death may also result from the selective degradation of specific survival-promoting or death-inhibiting factors. In this regard, it was recently shown that the caspase-inhibitor zVAD results in targeted autophagic degradation of catalase, leading to accumulation of radical oxygen species and death (Scott, 2007).
In summary, these results demonstrate that constitutive induction of autophagy inhibits cell growth and leads to cell death, highlighting the importance of physiological mechanisms that restrain this process. In addition, the finding that Atg1 expression is sufficient to induce autophagy provides a new tool for experimental or therapeutic manipulation of autophagy. Compounds that target the kinase activity of Atg1 may lead to novel therapies for the wide range of diseases linked to autophagy (Scott, 2007).
Selective macroautophagy is an important protective mechanism against diverse cellular stresses. In contrast to the well-characterized starvation-induced autophagy, the regulation of selective autophagy is largely unknown. This study demonstrates that Huntingtin, the Huntington disease gene product, functions as a scaffold protein for selective macroautophagy but is dispensable for non-selective macroautophagy. In Drosophila, Huntingtin genetically interacts with autophagy pathway components. In mammalian cells, Huntingtin physically interacts with the autophagy cargo receptor p62 to facilitate its association with the integral autophagosome component LC3 and with Lys-63-linked ubiquitin-modified substrates. Maximal activation of selective autophagy during stress was attained by the ability of Huntingtin to bind ULK1, a kinase that initiates autophagy, which released ULK1 from negative regulation by mTOR. This data uncovers an important physiological function of Huntingtin and provides a missing link in the activation of selective macroautophagy in metazoans (Rui, 2015).
Homozygous flies lacking the single htt homologue (dhttko) are fully viable with only mild phenotypes. In a genetic screen for the physiological function of Htt, ectopic expression of a truncated form of the microtubule-binding protein Tau (Tau-ΔC; truncated after Val 382) induced a prominent collapse of the thorax in dhttko flies due to severe muscle loss not observed by Tau expression alone, and accelerated decline in mobility and lifespan. These phenotypes were fully rescued by the dhtt genomic rescue transgene (‘dhttRescue’), suggesting that dhtt protects against Tau-induced pathogenic effects (Rui, 2015).
Although heterozygous dhttko/+ flies expressing Tau
(ATau; dhttko/+) seem normal, removing a single copy
of the fly LC3 gene, atg8a
(atg8ad4 mutant), in these flies also induces a collapsed
thorax and muscle loss, which can be phenocopied by expressing Tau
in homozygous atg8ad4−/− flies alone. Four
additional components of the early steps of the autophagy pathway,
atg1 (ULK1), atg7
and an adaptor for the selective recognition of autophagic cargo,
also exhibit strong genetic interactions with dhtt.
Consistent with its pivotal role in autophagy initiation, loss of
atg1 induces the strongest defect, and Tau expression can
induce a mild muscle loss phenotype even in heterozygous null atg1Δ3d.
Collectively, these genetic interaction studies suggest a role for
dhtt in autophagy (Rui, 2015).
By using the mCherry–GFP–Atg8a fusion reporter to directly measure autophagic flux in adult dhttko−/− brains, this study found similar number of red fluorescent punctae (acidic autolysosomes originating from autophagosome/lysosome fusion) in young mutant and control flies, but the number of punctae were reduced in old dhttko−/− brains when compared with age-matched controls. As autophagosome accumulation (co-localized green and red puncta) was not observed, it was concluded that the absence of dhtt in older animals was associated with reduced autophagosome formation. The fact that levels of Ref(2)P are significantly higher in old dhttko−/− brains compared with brains from age-matched wild-type controls suggests a possible preferential compromise in selective autophagy in these animals (Rui, 2015).
Consistent with the role of basal autophagy in quality control in non-dividing cells, it was found that brains from 5-week-old dhttko−/− contained almost double the amount of ubiquitylated proteins, a marker of quality control failure, compared with wild-type flies. As genetic interaction analysis and specific ubiquitin proteasome system (UPS) reporters all failed to reveal a functional link between dhtt and the UPS pathway, the study proposes that the defects in autophagic activity are the main cause of diminished quality control and increased accumulation of ubiquitylated proteins in dhttko−/− mutants (Rui, 2015).
Selective autophagy is induced in response to proteotoxic stress. The truncated Tau-ΔC used in genetic experiments in this study is preferentially degraded through autophagy in cortical neurons, serving as a model of proteotoxicity when ectopically expressed. The lower stability of Tau-ΔC compared with full-length Tau in wild-type flies and in UPS mutants was confirmed, but significantly higher levels of Tau-ΔC when expressed in atg8a and in dhttko−/− mutant flies were found, suggesting that autophagy is essential for the clearance of Tau-ΔC also in flies and that dhtt plays a role in this clearance (Rui, 2015).
In contrast, loss of dhtt does not affect flies’ adaptation to nutrient deprivation, which typically induces robust ‘in bulk’ autophagy. Fat bodies of early third instar larvae expressing mCherry–Atg8, where starvation-induced autophagy can be readily detected, fail to reveal any significant difference between wild-type and dhttko−/− flies; these flies die at the same rate as wild-type flies when tested for starvation resistance. Thus, although dhtt is necessary for selective autophagy of toxic proteins such as Tau-ΔC, it is dispensable for starvation-induced autophagy in flies (Rui, 2015).
Expression of human Htt (hHTT) in dhttko−/− null flies rescues both the mobility and longevity defects of dhttko−/− mutants and partially rescues the Tau-induced morphological and behavioural defects of dhttko−/− flies. hHTT also suppresses almost all of the autophagic defects observed in dhttko−/−, including decreased levels of autolysosomes, increased levels of Ref(2)P and of total ubiquitylated proteins, and accumulation of ectopically expressed Tau-ΔC, suggesting that the involvement of dhtt in autophagy is functionally conserved. In fact, confluent mouse fibroblasts knocked down for Htt (Htt(−)) exhibit significantly lower basal rates of long-lived proteins’ degradation than control cells, which are no longer evident on chemical inhibition of lysosomal proteolysis or of macroautophagy, thus confirming an autophagic origin of the proteolytic defect. Htt(−) fibroblasts also exhibit higher p62 levels and accumulate ubiquitin aggregates even in the absence of a proteotoxic challenge. As in dhttko−/− flies, Htt knockdown in mammalian cells does not affect degradation of CL1–GFP (a UPS reporter), β-catenin (a UPS canonical substrate) or proteasome peptidase activities. Reduced autophagic degradation in Htt(−) cells is not due to a primary lysosomal defect, as depletion of Htt does not reduce lysosomal acidification, endolysosomal number (if anything, an expansion of this compartment was observed) or other lysosomal functions such as endocytosis (for example, transferrin internalization). In fact, analysis of the lysosomal degradation of LC3-II reveals that autophagic flux and autophagosome formation are preserved and even enhanced in Htt(−) fibroblasts at basal conditions (Rui, 2015).
Autophagy is an evolutionary conserved process by which eukaryotic cells undergo self-digestion of cytoplasmic components. This study reports that a novel Drosophila immunophilin, named Zonda (CG5482), is critically required for starvation-induced autophagy. Zonda operates at early stages of the process, specifically for Vps34-mediated phosphatidylinositol 3-phosphate (PI3P) deposition. Zonda displays an even distribution under basal conditions, and soon after starvation nucleates in endoplasmic reticulum-associated foci that colocalize with omegasome markers. Zonda nucleation depends on Atg1, Atg13 and Atg17 but does not require Vps34, Vps15, Atg6 or Atg14. Zonda interacts physically with ATG1 through its kinase domain, as well as with ATG6 and Vps34. It is proposed that Zonda is an early component of the autophagy cascade necessary for Vps34-dependent PI3P deposition and omegasome formation (Melani, 2017).
Autophagy, one of the main degradative pathways of the cell, begins with the formation of a membranous cistern called phagophore or isolation membrane that buds from a cup-shaped structure associated with the endoplasmic reticulum (ER) called omegasome. Thereafter, the phagophore expands and finally seals, giving rise to a double membrane organelle named autophagosome where cytoplasmic components including protein aggregates, ribosomes, and mitochondria are sequestered. Soon afterward, autophagosomes acquire degradative enzymes by successive fusion with late endosomes and lysosomes, thereby becoming an autophagolysosome where the engulfed material is degraded (Melani, 2017).
Autophagy, whose main stimulus is the stress generated by nutrient deprivation, is modulated by intracellular signaling pathways, mainly the target of rapamycin (TOR) and AMP-activated protein kinase (AMPK) cascades, as well as by extracellular factors including hormones. Activation of the ULK1 complex (Atg1 complex in yeast and Drosophila) has been described as the first event in the autophagy cascade. This complex, formed by ULK1/2, FIP200/Atg17, Atg13, and Atg101, is constitutively assembled, and its kinase activity is negatively regulated by TOR signaling, which in turn depends on amino acid availability and the energy status of the cell. ULK1/Atg1 regulates the recruitment and activation of a second complex: the Vps34 lipid kinase complex, also called the autophagy nucleation complex, which is composed of the class 3 phosphatidylinositol 3-kinase Vps34 and the proteins PI3KR4 (Vps15), Beclin1 (BECN1)/Atg6, and Atg14 (Melani, 2017).
Vps34 mediates the synthesis of phosphatidylinositol 3-phosphate (PI3P). Local synthesis of this lipid defines the location of omegasome formation and, therefore the site of recruitment of several FYVE domain-containing proteins including DFCP1 and WIPI1, which in turn mediate phagophore elongation and autophagosome formation. Within the Vps34 complex, BECN1 is a direct target of ULK1/Atg1, and Vps34 kinase activity is believed to depend on the differential interaction of BECN1 with AMBRA1 or with the anti-apoptotic protein BCL-2. BCL-2 binding modulates the levels of BECN1 that become available to interact with Vps34 in the autophagy nucleation complex, thereby contributing to define if the cell will enter apoptosis or activate autophagy (Melani, 2017).
FK506-binding proteins (FKBPs) play a role in immunoregulation and participate in critical cellular functions that include protein trafficking and folding. Members of this family display peptidyl prolyl cis/trans isomerase (PPIase) activity, participating in de novo protein folding through the interconversion of intermediate folding states into the final tridimensional structure. This study has investigated a novel Drosophila gene-which has been named Zonda (Zda)-that encodes an immunophilin of the FKBP family, presumably homologous to mammalian FKBP8/FKBP38 (Bhujabal, 2017; Melani, 2017 and references therein).
By utilizing an in vivo approach, this study found that Zda is critically required for starvation-induced autophagy. Zda protein displays a cytoplasmic distribution in well-fed larvae and, shortly after the onset of starvation, nucleates in foci that colocalize with omegasome markers. Genetic manipulations revealed that components of the induction complex, Atg1, Atg13, and Atg17, but not components of the Vps34 complex, Vps34, Vps15, Atg6, or Atg14, are required for starvation-induced Zda nucleation. Moreover, Zda interacts physically with Atg1, Atg6, and Vps34 and is necessary for autophagic activation of Vps34 and omegasome formation, as revealed by DFCP1 foci formation following starvation. Zonda overexpression is sufficient to trigger a bona fide autophagic response, as evaluated by different autophagic markers. It is proposed that Zda is a novel component of the Drosophila autophagy machinery that forms part of the omegasome and is required for deposition of PI3P by the Vps34 complex and, hence, for the initiation of autophagosome biogenesis (Melani, 2017).
Previously, other immunophilins have been proposed both as positive or negative regulators of autophagy. In Drosophila, FKBP39 was found to be a negative regulator of developmentally triggered autophagy, possibly through the regulation of the transcription factor Foxo. Mammalian FKBP51 was described as a scaffold protein that recruits PHLPP, Akt, and Beclin1, leading to activation of autophagy. FKBP38 has been reported as a mitophagy receptor that interacts with LC3. Coexpression of FKBP38 along with LC3 can trigger Parkin-independent mitophagy (Melani, 2017).
Based on sequence homology, Zda is the likely orthologue of FKBP38. Not only do they share characteristics domains of FKBP proteins, but both proteins are the only members of their families to have a transmembrane domain on their C-terminal end. This study has shown that Zda is required for starvation-induced autophagy. Larval fat body cells in which Zda expression has been silenced fail to trigger autophagy, as assessed by several independent criteria: 1) inability of the cells to form autophagosomes and autolysosomes after starvation, as assessed by TEM and Atg8 nucleation; 2) their inability to increase the number and size of lysosomes, as evaluated by LysoTracker and GFP-Lamp markers; and 3) accumulation of Ref(2)P in these cells, which is indicative of impaired autophagic flux (Melani, 2017).
This study has found that, after nutrient deprivation, Zda can be detected in omegasomes, colocalizing with PI3P and DFCP1, from which early autophagic structures labeled with GFP-Atg5 and GFP-Atg8 bud off. Consistent with the notion that Zda is an early component of the autophagy cascade, genetic analysis revealed that starvation-induced Zda nucleation depends fully on components of the Atg1 induction complex but not on components of the Vps34 nucleation complex. Vps34 autophagic activation following starvation is regulated by the nutritional status of the cell downstream of Atg1. This study found that Zda interacts physically with the Atg1 kinase domain, as well as with components of the nucleation complex, including Atg6 and Vps34, suggesting that it may contribute to the activation of the latter complex by Atg1. This notion is consistent with the results of genetic experiments utilizing early autophagy markers, as they suggest that autophagy-dependent Vps34 activation and omegasome formation are dependent on Zda, this dependence being comparable to that on Atg1. Unlike Atg6, which was shown to be also required for Vps34 basal activity, Zda is clearly not necessary for early endosome formation but only for autophagic activation of Vps34. Thus, given the requirement of Zda for Vps34 autophagy-specific activation, and based on its localization at the omegasome, it is proposed that Zda contributes to define the location on the ER at which Vps34-dependent PI3P deposition and omegasome formation take place (Melani, 2017).
Induction of autophagy depends on the nutritional status of the cell and is subject to a contra-regulatory mechanism that occurs between mTOR and Atg1. Under nutrient-rich conditions, active mTOR phosphorylates and inactivates the Atg1 complex, and when nutrients are scarce, mTOR-dependent inactivation of Atg1 is released. Atg1 in turn reinforces down-regulation of mTOR through mechanisms that remain poorly defined. In line with this, Drosophila fat body cells that are mutant for atg1 grow bigger than control cells when subjected to prolonged nutrient deprivation, and conversely, Atg1 overexpression provokes cell size reduction and induces autophagosome formation. This study has shown that when overexpressed above certain levels, Zda can trigger a bona fide autophagic process, as assessed by several indicators, including TEM, Atg8 nucleation, and LysoTracker incorporation. This autophagic response fully depends on the activity of Vps34 and partially on Atg1. This suggests that Zonda operates upstream of Vps34 and in parallel to Atg1. Consistent with this, it was observed that under the same overexpression conditions, the TOR pathway is down-regulated and cell size is reduced similarly to what has been reported for Atg1. In line with these observations, adult flies that are homozygous for a Zda null mutation specifically in the head exhibit larger heads. Thus Zda mediates negative regulation of TOR, thereby exerting cell-autonomous negative regulation of growth (Melani, 2017).
Given that immunophilins are known to work as chaperons or scaffolds, it is proposed that Zda might provide a platform where Atg1 and the Vps34 complex interact. Further research is required to define the mechanism by which Zda cooperates with Atg1 on the activation of the Vps34 nucleation complex that culminates in localized PI3P deposition for omegasome formation (Melani, 2017).
Mutations in the human LMNA gene cause a collection of diseases known as laminopathies. These include myocardial diseases that exhibit age-dependent penetrance of dysrhythmias and heart failure. The LMNA gene encodes A-type lamins, intermediate filaments that support nuclear structure and organize the genome. Mechanisms by which mutant lamins cause age-dependent heart defects are not well understood. This study modeled human disease-causing mutations in the Drosophila Lamin C gene and expressed mutant Lamin C exclusively in the heart. This resulted in progressive cardiac dysfunction, loss of adipose tissue homeostasis, and a shortened adult lifespan. Within cardiac cells, mutant Lamin C aggregated in the cytoplasm, the CncC(Nrf2)/Keap1redox sensing pathway was activated, mitochondria exhibited abnormal morphology, and the autophagy cargo receptor Ref2(P)/p62 was upregulated. Simultaneous over-expression of the autophagy kinase Atg1 gene and an RNAi against CncC eliminated the cytoplasmic protein aggregates, restored cardiac function, and lengthened lifespan. These data suggest that simultaneously increasing rates of autophagy and blocking the Nrf2/Keap1 pathway are a potential therapeutic strategy for cardiac laminopathies (Bhide, 2018).
Mutations in the human LMNA gene are associated with a collection of diseases called laminopathies in which the most common manifestation is progressive cardiac disease. This study has generated Drosophila melanogaster models of age-dependent cardiac dysfunction. In these models, mutations synonymous with those causing disease in humans were introduced into Drosophila LamC. Cardiac-specific expression of mutant LamC resulted in (1) cardiac contractility, conduction, and physiological defects, (2) abnormal nuclear envelope morphology, (3) cytoplasmic LamC aggregation, (4) nuclear enrichment of the redox transcriptional regulator CncC (mammalian Nrf2), (5) and upregulation of autophagy cargo receptor Ref(2)P (mammalian p62). These cardiac defects were enhanced with age and accompanied by increased adipose tissue in the adult fat bodies and a shortened lifespan (Bhide, 2018).
To understand the mechanistic basis of cardiolaminopathy and identify genetic suppressors, advantage was taken of powerful genetic tools available in Drosophila. The presence of cytoplasmic LamC aggregates prompted a determination of whether increasing autophagy would suppress the cardiac defects. Cardiac-specific upregulation of autophagy (Atg1 OE) suppressed G489V-induced cardiac defects. Consistent with this, decreased autophagy due to expression of Atg1 DN resulted in enhanced deterioration of G489V-induced cardiac dysfunction. Interestingly, cardiac-specific Atg5 OE and Atg8a OE, two factors that also promote autophagy, showed little to no suppression of G489V-induced heart dysfunction, suggesting that Atg1 might be rate limiting in this context. These findings are consistent with studies in mouse laminopathy models in which rapamycin and temsirolimus had beneficial effects on heart and skeletal muscle through inhibition of AKT/mTOR signaling. These findings are depicted in a model (see Model for the interactions between the autophagy and CncC/Keap1 signaling pathway in mutant lamin-induced cardiac disease) in which cytoplasmic aggregation of mutant LamC results in upregulation of p62, which in turn inhibits autophagy via activation of TOR and inactivation of AMPK. AMPK inactivation leads to the activation of PI3K/Akt/mTOR pathway and inhibition of autophagy Atg1 OE promoted clearance of the LamC aggregates and restored proteostasis in these Drosophila models. Thus, the data suggest that mutant LamC reduces autophagy, resulting in impairment of cellular proteostasis that leads to cardiac dysfunction (Bhide, 2018).
Cardiac-specific expression of mutant LamC altered CncC subcellular localization. Previously, Drosophila larval body wall muscles expressing G489V were shown to experience reductive stress, an atypical redox state characterized by high levels of reduced glutathione and NADPH, and upregulation CncC target genes (Dialynas, 2015). Cardiac-specific CncC RNAi in the wild-type LamC background did not produce major cardiac defects. Consistent with this, Nrf2 deficiency in mice does not compromise cardiac and skeletal muscle performance. Cardiac-specific CncC RNAi suppressed G489V-induced cardiac dysfunction and reduced cytoplasmic LamC aggregation, but not R205W-induced defects. However, cardiac-specific RNAi against CncC did not affect G489V-induced adipose tissue accumulation and lifespan shortening. Similar to the nuclear enrichment of CncC in hearts expressing G489V, human muscle biopsy tissue from an individual with a point mutation in the LMNA gene that results in G449V (analogous to Drosophila G489V) showed nuclear enrichment of Nrf2 (Dialynas, 2015). Disruption of Nrf2/Keap1 signaling has also been reported for Hutchinson-Gilford progeria, an early-onset aging disease caused by mutations in LMNA. In this case, however, the thickened nuclear lamina traps Nrf2 at the nuclear envelope that results in a failure to activate Nrf2 target genes, leading to oxidative stress. In these studies, CncC nuclear enrichment was observed; however, a redox imbalance was not readily observed at the three-time points investigated. This might indicate that there is a window of time in disease progression in which redox imbalance occurs and that mechanisms are in place to re-establish homeostasis (Bhide, 2018).
It has been postulated that there is cross-talk between autophagy and Nrf2/Keap1 signaling. This was tested by manipulating autophagy and CncC (Nrf2) alone and in combination. CncC RNAi suppressed the cardiac defects caused by G489V, but not the lipid accumulation and lifespan shortening, suggesting the latter two phenotypes are not specifically due to loss of cardiac function. In contrast, Atg1 OE suppressed the cardiac and adipose tissue defects and lengthened the lifespan. The double treatment (simultaneous Atg1 OE and RNAi knockdown of CncC) gave the most robust suppression of the mutant phenotypes and completely restored the lifespan. Interestingly, Atg1 DN and RNAi knockdown of CncC simultaneously did not further deteriorate or improve the mutant phenotypes. Taken together, these data suggest that autophagy plays a key role in suppression of the G498V-induced phenotypes and that knockdown on CncC enhances this suppression (Bhide, 2018).
These findings support a model whereby autophagy and Nrf2 signaling are central to cardiac health. It is proposed that cytoplasmic aggregation of LamC increases levels of Ref(2)P (p62), which competitively binds to Keap1, resulting in CncC (Nrf2) translocation to the nucleus. Inside the nucleus, Nrf2 regulates genes involved in detoxification. Continued expression of antioxidant genes results in the disruption of redox homeostasis, defective mitochondria, and dysregulation of energy homeostasis/energy sensor such as AMPK and its downstream targets. Simultaneously, upregulation of Ref(2)P (p62) causes inhibition of autophagy via activation of TOR, which leads to the inactivation of AMPK. AMPK inactivation in combination with activation of the TOR pathway causes cellular and metabolic stress that leads to cardiomyopathy. In support of this model, transcriptomics data from muscle tissue of an individual with muscular dystrophy expressing Lamin A/C G449V (analogous to Drosophila G489V) showed (1) upregulation of transcripts from Nrf2 target genes, (2) upregulation of genes encoding subunits of the mTOR complex, and (3) downregulation of AMPK, further demonstrating relevance of the Drosophila model for providing insights on human pathology (Bhide, 2018).
AMPK exerts prolongevity effects in diverse species; however, the tissue-specific mechanisms involved are poorly understood. This study shows that upregulation of AMPK in the adult Drosophila nervous system induces autophagy both in the brain and also in the intestinal epithelium. Induction of autophagy is linked to improved intestinal homeostasis during aging and extended lifespan. Neuronal upregulation of the autophagy-specific protein kinase Atg1 is both necessary and sufficient to induce these intertissue effects during aging and to prolong the lifespan. Furthermore, upregulation of AMPK in the adult intestine induces autophagy both cell autonomously and non-cell-autonomously in the brain, slows systemic aging, and prolongs the lifespan. The organism-wide response to tissue-specific AMPK/Atg1 activation is linked to reduced insulin-like peptide levels in the brain and a systemic increase in 4E-BP expression. Together, these results reveal that localized activation of AMPK and/or Atg1 in key tissues can slow aging in a non-cell-autonomous manner (Ulgherait, 2014. PubMed ID: 25199830).
The TOR kinases are conserved negative regulators of autophagy in response to nutrient conditions, but the signaling mechanisms are poorly understood. This study describes a complex containing the protein kinase Atg1 and the phosphoprotein Atg13 that functions as a critical component of this regulation in Drosophila. Knockout of Atg1 or Atg13 results in a similar, selective defect in autophagy in response to TOR inactivation. Atg1 physically interacts with TOR and Atg13 in vivo, and both Atg1 and Atg13 are phosphorylated in a nutrient-, TOR- and Atg1 kinase-dependent manner. In contrast to yeast, phosphorylation of Atg13 is greatest under autophagic conditions and does not preclude Atg1-Atg13 association. Atg13 stimulates both the autophagic activity of Atg1 and its inhibition of cell growth and TOR signaling, in part by disrupting the normal trafficking of TOR. In contrast to the effects of normal Atg13 levels, increased expression of Atg13 inhibits autophagosome expansion and recruitment of Atg8/LC3, potentially by decreasing the stability of Atg1 and facilitating its inhibitory phosphorylation by TOR. Atg1/Atg13 complexes thus function at multiple levels to mediate and adjust nutrient-dependent autophagic signaling (Chang, 2009).
The remarkable conservation of autophagy-related proteins and processes between yeast and higher eukaryotes has contributed to a rapid advance in understanding of this process in multicellular animals. The data presented in this study extend this conservation to the mechanisms of autophagy regulation by nutrient-dependent TOR signaling. As in yeast, Drosophila Atg13 forms a complex with Atg1, stimulates its activity and is highly phosphorylated in a nutrient and TOR-dependent manner. Although the relevant substrates of Atg1 remain to be identified, these results suggest that this fundamental pathway from nutrient signal to autophagic induction has been largely retained between yeast and metazoans. This regulatory link between TOR and Atg1/Atg13 thus represents one of the most highly conserved TOR outputs described to date. Despite this conservation, critical differences were identified in the behaviors of these proteins, which likely stem in part from differences in the physiology of lower vs. higher eukaryotes. Chan (2009) reported an essential role in autophagy for human Atg13 using RNAi-mediated knockdown in cultured cell lines, and described interactions between Atg13 and Ulk1 and Ulk2 similar to those reported in this study. Hosokawa (2009) and Jung (2009) also describe related interactions between mammalian Atg13 and Ulk kinases. Together, these studies point to a model whereby Atg13 is phosphorylated and interacts with Atg1 under both fed and starved conditions in metazoans, in contrast to the growth-dependent phosphorylation and dissociation of Atg13/Atg1 observed in yeast. Although the greater nutrient buffering capacity of multi- vs. unicellular animals probably contributes to these differences, the requirement for significant basal rates of autophagy in the maintenance of large, long-lived metazoan cells may dictate that the Atg1-Atg13 complex remain partially active even under fed conditions. Furthermore, although Atg1 can clearly associate with phosphorylated Atg13, dephosphorylation of TOR-dependent sites on Atg13 may be masked by increased phosphorylation by Atg1, but may nonetheless contribute to regulation of Atg1-Atg13 interaction or activity. The observation that Atg1 kinase activity plays a role under both fed and starved conditions in supporting hyperphosphorylation of Atg13 suggests that rather than switching Atg1 kinase between off and on states, nutrient signals may instead affect its substrate specificity or accessibility (Chang, 2009).
Metazoan Atg1/Atg13 complexes have also accrued additional regulatory mechanisms that have not been described in yeast, including negative feedback from Atg1 to TOR, phosphorylation of Atg13 by Atg1, and nutrient-dependent effects on the stability of these proteins. These additional layers of regulation are in keeping with the elaboration of intracellular signaling pathways in metazoans. The observation that Atg13 phosphorylation is both TOR- and Atg1-dependent under fed conditions suggests a model whereby phosphorylation by one of these kinases may serve as a priming event for the other. In addition, the role of Atg1-dependent phosphorylation of Atg13 under starvation conditions remains an important question. Whereas the Atg1-independent localization of Atg13 to autophagosomes and its activation of Atg1 indicate that Atg13 functions upstream of Atg1, the finding that Atg13 acts as a substrate for Atg1-dependent phosphorylation raises the possibility that Atg13 may also act to transduce signals downstream of Atg1. Identification of additional Atg1 substrates and Atg13 interacting proteins may help clarify this issue (Chang, 2009).
Given its positive role in autophagy induction, the ability of Atg13 to inhibit autophagy when overexpressed was unexpected. This may in part reflect a dominant negative effect of Atg13 overexpression causing titration of Atg1 complexes or competition with other Atg1 substrates. However, the observation that Atg13 stimulates TOR-dependent phosphorylation of Atg1 and that Atg1 levels increase in Atg13 mutant cells suggests that Atg13 has both positive and negative roles in autophagy induction. These opposing activities of Atg13 are reminiscent of the mTOR complex I component Raptor, which plays an essential role in TOR signaling yet also inhibits TOR activity under starvation conditions. The results suggest that Atg13 may play an analogous role, switching between states of promoting or inhibiting autophagy, thereby sharpening the response to changes in nutrient conditions. These findings suggest that the relative ratio of Atg13 to Atg1 or to other components of the complex may play an important role in dictating its activity. In this regard, the differential regulation of Atg1 and Atg13 levels by TOR could provide an additional mechanism whereby TOR signaling can influence autophagic activity. Whereas the autophagy-defective phenotypes of Atg1 and Atg13 mutants reveals the dominant positive roles of these genes, disruption of other putative components of this complex leads to autophagy induction, suggesting some components may play primarily negative roles (Chang, 2009).
In conclusion, the results demonstrate that Atg1/Atg13 complexes play an essential, conserved role in promoting autophagy in response to TOR inactivation, with additional regulatory functions unique to metazoans. Further insight into the regulation of this complex and identification of its targets may lead to additional means of manipulating autophagy rates for therapeutic purposes (Chang, 2009).
Autophagy is a physiological and evolutionarily conserved process maintaining homeostatic functions, such as protein degradation and organelle turnover. Accumulating data provide evidence that autophagy also contributes to cell death under certain circumstances, but how this is achieved is not well known. This study reports that autophagy occurs during developmentally-induced cell death in the female germline, observed in the germarium and during middle developmental stages of oogenesis in Drosophila. Degenerating germline cells exhibit caspase activation, chromatin condensation, DNA fragmentation and punctate staining of mCherry-DrAtg8a, a novel marker for monitoring autophagy in Drosophila. Genetic inhibition of autophagy, by removing atg1 or atg7 function, results in significant reduction of DNA fragmentation, suggesting that autophagy acts genetically upstream of DNA fragmentation in this tissue. This study provides new insights into the mechanisms that regulate cell death in vivo during development (Nezis, 2009).
The TUNEK assay was used ti measure cell death during Drosophila oogenesis. Such analysis revealed that cell death in the germarium occurred in 26% of the ovarioles, in young, well-fed flies. In contrast, the percentage of cell death during middle stages of oogenesis was 9.7% under the same conditions. In the germarium the degenerated germline cells are usually located in region 2 and, during middle stages of oogenesis, in egg chambers of developmental stages 7, 8 and 9. In order to detect caspase activity, immunolabeling was performed with the anti-active caspase-3 (cleaved caspase-3) antibody. The degenerated germaria revealed an intense staining in region 2, indicating the presence of activated caspase-3 proteases and therefore caspase-dependent cell death. A similar staining pattern is observed in the degenerating mid-stage egg chambers. Next whether autophagy occurs during cell death in the germarium and mid-stage egg chambers of Drosophila was investigated. GFP-tagged Atg8a or its vertebrate homologue LC3, has been extensively used to monitor autophagy as it labels the autophagic membranes. However, the pH sensitivity of GFP makes it impossible to follow GFP-Atg8a after the short-lived autophagosomes fuse with lysosomes. Fusion with late endosomes to create amphisomes may also lead to an environment where the fluorescence from GFP is quenched due to low pH. Therefore a transgenic fly was made in which DrAtg8a is fused to the acid-insensitive fluorescent protein mCherry that can be easily monitored into amphisomes and autolysosomes. Upon expression of mCherry-DrAtg8a in the germline using the germline-specific driver, nanos-VP16-Gal4 several mCherry-DrAtg8a puncta were detected in region 2 of the germarium and in the degenerating mid-stage egg chambers, indicating autophagic activity. When mCherry-DrAtg8a was coexpressed with GFP-LC3, it was observed that the number of mCherry-positive structures in degenerating mid-stage egg chambers was significantly higher compared to the number of GFP-positive structures. This observation reveals that mCherry-DrAtg8a is a very useful marker for monitoring autophagy in Drosophila, since it can be used for detecting both autophagosomes and autolysosomes (Nezis, 2009).
Examination of the ultrastructural morphology of degenerated germaria revealed that the cytoplasm of the germline cells indeed contained a variety of autophagosomes and autolysosomes. Autophagic compartments were filled with cytoplasm, numerous vesicles, dense masses and multi-lamellar membranes of various sizes. Additionally, while the nuclei in the normal non-degenerating germ line cysts were very irregular in shape with many invaginations and protrusions, the nuclei in the degenerated cysts had a round shape and condensed chromatin. Surprisingly, all these nuclei had irregular and extensive nuclear membrane dilation. Together, the above data demonstrate that autophagy occurs in caspase-dependent cell death during early oogenesis in Drosophila (Nezis, 2009).
To explore the potential role of autophagy in the cell death process of the germline cells in the germarium and in mid-stage egg chambers, germline mutant cells were generated for the core Drosophila autophagy genes atg1 and atg7 and scored for apoptotic cell death using the TUNEL assay in order to detect DNA fragmentation. Interestingly, cell death in the germarium of atg1 germline mutants was reduced to 8.4% (compared to 26% of the control) and to 3.5% for mid-stage egg chambers. atg7 germline mutants exhibited a similar reduction in apoptotic cell death. The percentage of germaria being positive for TUNEL was 13.8% and the percentage of midstage egg chambers being positive for TUNEL was 4.8%. The above data indicate that inhibition of autophagy reduces DNA fragmentation, suggesting that during Drosophila early oogenesis autophagy acts upstream of caspase activation and DNA fragmentation (Nezis, 2009).
How can autophagy promote cell death by acting upstream of caspase activation? One possible explanation is that proteins crucial for cell survival and organelles are degraded by autophagy, thus promoting cell death. Moreover, autophagy could also act in parallel and cooperatively with caspases for the most efficient degradation of the tissue. In future studies, molecular mechanisms that illuminate the contribution of autophagy in the cell death process will be identified (Nezis, 2009).
Autophagy is an evolutionarily conserved pathway responsible for degradation of cytoplasmic material via the lysosome. Although autophagy has been reported to contribute to cell death, the underlying mechanisms remain largely unknown. This study shows that autophagy controls DNA fragmentation during late oogenesis in Drosophila. Inhibition of autophagy by genetically removing the function of the autophagy genes atg1, atg13, and vps34 resulted in late stage egg chambers that contained persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. The Drosophila inhibitor of apoptosis (IAP) dBruce was found to colocalize with the autophagic marker GFP-Atg8a and accumulated in autophagy mutants. Nurse cells lacking Atg1 or Vps34 in addition to dBruce contained persisting nurse cell nuclei with fragmented DNA. This indicates that autophagic degradation of dBruce controls DNA fragmentation in nurse cells. These results reveal autophagic degradation of an IAP as a novel mechanism of triggering cell death and thereby provide a mechanistic link between autophagy and cell death (Nezis, 2010).
Dying nurse cells exhibit several markers of apoptosis during late oogenesis in Drosophila such as caspase activation, chromatin condensation, and DNA fragmentation. To address the role of autophagy in nurse cell death, transgenic flies were generated carrying a UASp-GFP-mCherry-DrAtg8a transgene. The double-tagged Atg8a protein emits yellow (green merged with red) fluorescence in nonacidic structures such as autophagosomes, and is red only in the autolysosomes due to quenching of GFP in these acidic structures. Upon expression of GFP-mCherry-DrAtg8a in the germline, several GFP-mCherry-DrAtg8a yellow puncta were detected in the cytoplasm of nurse cells during early stage 12. After the completion of transport of the majority of the nurse cell cytoplasm to the growing oocyte during late stage 12, GFP-mCherry-DrAtg8a yellow puncta remained in nurse cell cytoplasm in close proximity to the nurse cell nuclei. Ultrastructural analysis of the nurse cells at the same developmental stage also revealed the presence of autophagosomes in the remaining nurse cell cytoplasm. Interestingly, during late stage 13 when the majority of nurse cells have degenerated, a large number of red structures were observed, indicating that the majority of the autophagosomes became autolysosomes. This was confirmed by ultrastructural analysis through detection of large autolysosomes associated with the condensed and fragmented nurse cell nucleus. These autolysosomes often contained condensed material resembling the material of the fragmented nurse cell nucleus, suggesting that the nurse cell nuclear remnants are removed by autophagy. Indeed, nurse cells of late stage 13 egg chambers expressing UASp-mCherry-DrAtg8a exhibited mCherry-DrAtg8a puncta that are located either adjacent to or attached to the fragmented nucleus, indicative of nuclear autophagy. To further examine the presence of autophagy during late oogenesis in Drosophila, protein trap lines were used that express GFP-tagged Atg5 and Atg8a. Atg5-GFP and Atg8a-GFP were detected as punctae around the nurse cell nuclei during late oogenesis, revealing the presence of autophagic compartments. These findings indicate that autophagy occurs during nurse cell death and degradation in late oogenesis in Drosophila (Nezis, 2010).
To explore the potential role of autophagy in nurse cell death during late oogenesis, germline mutant cells were generated for the core Drosophila autophagy genes atg1 and atg13 and cell death was examined using the TUNEL assay to detect fragmented DNA. Interestingly, in either atg1 or atg13 germline mutants, a significant increase was observed in the number of stage 14 egg chambers that had persisting TUNEL-negative nurse cell nuclei. This phenotype differs from wild-type stage 14 egg chambers, in which nurse cell nuclei can rarely be detected, and those few that remain are exclusively TUNEL positive. TUNEL-positive nurse cell nuclei can be detected in the wild-type egg chambers in earlier developmental stages but not in autophagy germline mutants. To further examine the role of autophagy in nurse cell degeneration, germline mutants were generated for vps34, a member of the class III PI3-kinase complex that is responsible for the production of phosphatidylinositol 3-phosphate, a phosphoinositide required for autophagy. Like the other autophagy mutants, the vps34 germline mutant egg chambers displayed significant increase in the number of egg chambers that had persisting TUNEL-negative nurse cell nuclei during late oogenesis. All autophagy germline mutants exhibited accumulation of Ref(2)P, a marker for autophagic flux, in the nurse cell cytoplasm compared with the wild type, further confirming that autophagy was inhibited. Interestingly, in all the autophagy germline mutants, the persisting nurse cell nuclei exhibited condensed nuclear staining. To examine whether proteolytic processing of caspase-3 was affected by inhibition of autophagy, immunolabeling for cleaved caspase-3 was performed in the atg1, atg13, and vps34 germline mutant egg chambers. Cleaved caspase-3 levels were markedly attenuated in autophagy germline mutants compared with the wild type, with 92% cleaved caspase-3 labeling in w1118 late stage 12-14 egg chambers, 38% in atg13−/− GLCs, and 33% in vps34−/− GLCs late stage 12-14 egg chambers. Together, these data demonstrate that autophagy functions upstream of caspase processing and DNA fragmentation during late oogenesis in Drosophila (Nezis, 2010).
How can autophagy promote caspase activity, DNA fragmentation, and cell death in the same cell? It was hypothesized that proteins crucial for cell survival could be degraded by autophagy, thus promoting cell death. To test this hypothesis, the localization of Drosophila IAPs in the nurse cells was investigated during late oogenesis along with their relationship to the autophagic marker GFP-Atg8a. Three of four known Drosophila IAPs, DIAP1, DIAP2, and dBruce were investigated. DIAP1 and DIAP2 exhibit a rather diffuse cytoplasmic staining that did not colocalize with GFP-Atg8a. In contrast, dBruce exhibited an interesting localization pattern. dBruce could not be detected in stage 10B egg chambers. Interestingly, during early stage 12, colocalization of dBruce and Atg8a-GFP was observed in structures 0.5-1.5 µm in diameter resembling autophagosomes. A similar pattern of colocalization was observed during late stage 12. In contrast, in later stages when nurse cell cytoplasm was completely transferred to the oocyte, dBruce exhibited a diffuse localization pattern mainly in the follicle cells surrounding the nurse cells remnants. These data suggest that dBruce might be degraded by autophagy. To test this hypothesis, the localization of dBruce was investigated in atg1, atg13, and vps34 germline mutants. Significantly, dBruce accumulated in the remaining cytoplasm of the nurse cells of all of these autophagy mutants and formed large aggregates 5-10 µm in diameter. Western blot analyses showed that autophagy germline mutant egg chambers contain higher levels of dBruce protein than wild-type egg chambers. These observations support the hypothesis and indicate that dBruce is degraded by autophagy in the nurse cells during late oogenesis (Nezis, 2010).
It was next asked how dBruce might be targeted for autophagy. p62 is a known adaptor protein that targets substrates for autophagic degradation. It was asked whether the Drosophila orthologue of p62, Ref(2)P may target dBruce for autophagy. Immunofluorescence analysis demonstrated that Ref(2)P staining in the nurse cells of late stage egg chambers has no correlation with the autophagic marker Atg8a-GFP. Additionally, Ref(2)P mutant egg chambers exhibited a normal pattern of DNA fragmentation, cell death, and degradation in the nurse cells during late oogenesis, which suggests that targeting dBruce for autophagy does not depend on Ref(2)P function (Nezis, 2010).
dBruce belongs to the IAP protein family. It contains both BIR (baculoviral IAP repeat, which is responsible for caspase inhibition) and UBC (responsible for ubiquitin conjugation) domains in the N and C termini, respectively. The function was tested of three different dBruce mutant alleles that result in truncated proteins with deletions either in the BIR or UBC domains. Two of them (dBruceE16 and dBrucee00984) have a deletion in the UBC domain, and one of them (dBruceE81) has a deletion in the BIR domain. All dBruce mutant alleles displayed a significant increase in the number of degenerating egg chambers during mid-oogenesis compared with the wild type. To further investigate the role of autophagic degradation of dBruce in nurse cell death, double mutants were constructed for either atg1 and dBruceE81 or vps34 and dBruceE81. Both double mutant egg chambers contained persistent nurse cell nuclei that were TUNEL positive. These data indicate that autophagic degradation of dBruce controls DNA fragmentation in the nurse cells during oogenesis in Drosophila (Nezis, 2010).
The role of autophagy in cell death has been controversial. Previous studies have shown that autophagy promotes cell death in Drosophila larval salivary glands, midgut, and embryonic serosal membrane. However, the precise mechanism by which autophagy executes the death of these cells is not clear. This study has shown that autophagic degradation of the IAP dBruce controls DNA fragmentation in nurse cells during Drosophila late oogenesis. The data also demonstrate that autophagy acts genetically upstream of caspase activation and DNA fragmentation in this developmental context and indicate that autophagy directly contributes to the activation of cell death. This agrees with recent evidence from cultured mammalian cells in which autophagy appears to act upstream of caspase-3 activation under specific experimental settings (Nezis, 2010 and references therein).
dBruce has been previously shown to suppress cell death in the Drosophila eye and also has a crucial function in nuclear degeneration during sperm differentiation in Drosophila. Interestingly, dBruce was recently shown to regulate autophagy and cell death during early and mid-oogenesis in Drosophila. In this earlier study, dBruce and caspase activity were shown to influence autophagy. In contrast, this study provides the first evidence for a mechanism by which autophagy regulates dBruce and cell death. This study provides genetic evidence that dBruce is degraded by autophagy in the degenerating nurse cells during late oogenesis and that it regulates DNA fragmentation. The fact that chromatin condensation is not affected in autophagy mutants indicates that this process is regulated independently from DNA fragmentation (Nezis, 2010),
Degradation of proteins that are crucial for cell survival is one of the mechanisms by which a cell can trigger its own death. For instance, selective depletion of catalase by autophagy has been shown to promote cell death in mammalian cells in vitro. Furthermore, it was recently shown that chaperone-mediated autophagy modulates the neuronal survival machinery by regulating the neuronal survival factor MEF2D, and dysregulation of this pathway is associated with Parkinson's disease. In a recent study, it was also demonstrated that autophagy promotes synaptogenesis in Drosophila neuromuscular junction by degrading Highwire, an E3 ubiquitin ligase which limits neuromuscular junction growth. The current in vivo data further support the idea that autophagic degradation of survival factors can promote cell death and indicate that IAPs can be degraded by autophagy, thereby causing cell death. Autophagy not only functions during late oogenesis as the cause of cell death, but can also function to efficiently degrade the nurse cell nuclei remnants, as previously shown in salivary glands. It was recently reported that dying nurse cells during late oogenesis exhibit characteristics of programmed necrosis and that the lysosomal genes dor, spinster, and cathepsin D are required for this process, showing that autophagy and necrosis participate in nurse cell death and degradation during late oogenesis. In conclusion, these findings indicate that autophagy plays an important role in nurse cell death during late oogenesis in Drosophila, first by acting upstream of DNA fragmentation, thereby causing cell death, and then by scavenging nurse cell remnants (Nezis, 2010).
Adult neurogenesis occurs in specific locations in the brains of many animals, including some insects, and relies on mitotic neural stem cells. In mammals, the regenerative capacity of most of the adult nervous system is extremely limited, possibly because of the absence of neural stem cells. This study shows that the absence of adult neurogenesis in Drosophila results from the elimination of neural stem cells (neuroblasts) during development. Prior to their elimination, their growth and proliferation slows because of decreased insulin/PI3 kinase signaling, resulting in nuclear localization of Foxo. These small neuroblasts are typically eliminated by caspase-dependent cell death, and not exclusively by terminal differentiation as has been proposed. Eliminating Foxo, together with inhibition of reaper family proapoptotic genes, promotes long-term survival of neuroblasts and sustains neurogenesis in the adult mushroom body (mb), the center for learning and memory in Drosophila. Foxo likely activates autophagic cell death, because simultaneous inhibition of ATG1 (autophagy-specific gene 1) and apoptosis also promotes long-term mb neuroblast survival. mb neurons generated in adults incorporate into the existing mb neuropil, suggesting that their identity and neuronal pathfinding cues are both intact. Thus, inhibition of the pathways that normally function to eliminate neural stem cells during development enables adult neurogenesis (Siegrist, 2010).
These findings demonstrate that two pathways act in concert to eliminate mb neuroblasts and terminate neurogenesis. Downregulation of insulin/PI3 kinase signaling occurs first and may activate both autophagy and a program of caspase-dependent cell death. In the absence of one of these cell death pathways, mb neuroblasts persist, but only transiently. Thus a fail-safe mechanism likely exits to ensure mb neuroblast elimination, similar to salivary gland cells (Siegrist, 2010).
The reduction in growth that precedes neuroblast apoptosis appears to be developmentally regulated since it occurs at an earlier time in central brain neuroblasts than in mushroom body neuroblasts. This may be due to either local differences in microenvironments or differences in the ability of neuroblasts to respond to circulating insulin-like peptides. Moreover, the extended survival of mb neuroblasts under these conditions, but not other central brain neuroblasts, suggests that additional mechanisms such as terminal differentiation still function to ensure elimination of most neuroblasts. Indeed, during mammalian development, neural progenitors are eliminated via cell death and by terminal differentiation. The relative importance of death and differentiation for neuroblast elimination may be lineage dependent. Finally because cricket adult mb neuroblasts proliferate in response to insulin in explant cultures, a common mechanism may regulate adult neurogenesis among insects and possibly in more distantly related metazoans. These findings may represent an important first step towards devising ways to manipulate the regenerative capacity of adult brains in diverse species and provide insight into how aberrantly persisting neural stem cells behave in vivo (Siegrist, 2010).
Phagophore-derived autophagosomes deliver cytoplasmic material to lysosomes for degradation and reuse. Autophagy mediated by the incompletely characterized actions of Atg proteins is involved in numerous physiological and pathological settings including stress resistance, immunity, aging, cancer, and neurodegenerative diseases. This study characterized tg17/FIP200A, the Drosophila ortholog of mammalian RB1CC1/FIP200, a proposed functional equivalent of yeast Atg17. Atg17 disruption inhibits basal, starvation-induced and developmental autophagy, and interferes with the programmed elimination of larval salivary glands and midgut during metamorphosis. Upon starvation, Atg17-positive structures appear at aggregates of the selective cargo Ref(2)P/p62 near lysosomes. This location may be similar to the perivacuolar PAS (phagophore assembly site) described in yeast. Drosophila Atg17 is a member of the Atg1 kinase complex as in mammals, and it binds to the other subunits including Atg1, Atg13 and Atg101 (C12orf44 in humans, 9430023L20Rik in mice and RGD1359310 in rats). Atg17 is required for the kinase activity of endogenous Atg1 in vivo, as loss of Atg17 prevents the Atg1-dependent shift of endogenous Atg13 to hyperphosphorylated forms, and also blocks punctate Atg1 localization during starvation. Finally, it was found that Atg1 overexpression induces autophagy and reduces cell size in Atg17-null mutant fat body cells, and that overexpression of Atg17 promotes endogenous Atg13 phosphorylation and enhances autophagy in an Atg1-dependent manner in the fat body. A model is proposed according to which the relative activity of Atg1, estimated by the ratio of hyper- to hypophosphorylated Atg13, contributes to setting low (basal) vs. high (starvation-induced) autophagy levels in Drosophila (Nagy, 2014).
The selective degeneration of dendrites precedes neuronal cell death in hypoxia-ischemia (HI) and is a neuropathological hallmark of stroke. While it is clear that a number of different molecular pathways likely contribute to neuronal cell death in HI, the mechanisms that govern HI-induced dendrite degeneration are largely unknown. This study shows that the NAD synthase nicotinamide mononucleotide adenylyltransferase (Nmnat) functions endogenously to protect Drosophila class IV dendritic arborization (da) sensory neurons against hypoxia-induced dendritic damage. Whereas dendrites of wild-type class IV neurons are largely resistant to morphological changes during prolonged periods of hypoxia, class IV neurons of nmnat heterozygous mutants exhibit significant dendrite loss and extensive fragmentation of the dendritic arbor under the same hypoxic conditions. Although basal levels of autophagy are required for neuronal survival, this study demonstrates that autophagy is dispensable for maintaining the dendritic integrity of class IV neurons. However, it was found that genetically blocking autophagy can suppress hypoxia-induced dendrite degeneration of nmnat heterozygous mutants in a cell-autonomous manner, suggestive of a self-destructive role for autophagy in this context. It was further shown that inducing autophagy by overexpression of the autophagy-specific kinase Atg1 is sufficient to cause dendrite degeneration of class IV neurons under hypoxia and that overexpression of Nmnat fails to protect class IV dendrites from the effects of Atg1 overexpression. These studies reveal an essential neuroprotective role for endogenous Nmnat in hypoxia and demonstrate that Nmnat functions upstream of autophagy to mitigate the damage incurred by dendrites in neurons under hypoxic stress. These studies further support the possibility that Nmnat protects against dendrite degeneration by maintaining low levels of ROS or by attenuating the deleterious effects of accumulated ROS induced by hypoxia (Wen, 2013).
Autophagy is a conserved cellular process of macromolecule recycling that involves vesicle-mediated degradation of cytoplasmic components. Autophagy plays essential roles in normal cell homeostasis and development, the response to stresses such as nutrient starvation, and contributes to disease processes including cancer and neurodegeneration. Although many of the autophagy components identified from genetic screens in yeast are well conserved in higher organisms, the mechanisms by which this process is regulated in any species are just beginning to be elucidated. In a genetic screen in Drosophila melanogaster, a link was identified between the focal adhesion protein paxillin and the Atg1 kinase, which has been previously implicated in autophagy. In mammalian cells, paxillin was found to redistributed from focal adhesions during nutrient deprivation, and paxillin-deficient cells exhibit defects in autophagosome formation. Together, these findings reveal a novel evolutionarily conserved role for paxillin in autophagy (Chen, 2008).
This paper reports genetic interactions between paxillin and components implicated in developmental autophagy, including Atg1 and the ecdysone receptor, during wing maturation. Intriguingly, in te newly emerged adults, autophagy is also observed at the time of wing spreading. It was also found that over-expression of DSRF, which suppresses Pax-induced wing blisters, rescues Atg1-induced wing defects, thus further supporting a role of Atg1 in wing morphogenesis. EP3348, which strongly suppresses Pax-induced blistered wings, harbors a single transposable element inserted in the 5' untranslated region of the Drosophila Atg1 gene. It is notable that EP3348 suppresses Pax-induced wing defects more effectively than does Atg1-RNAi. Since Atg1 mutants exhibit a pupal lethal phenotype, but neither EP3348 nor expression of Atg1-RNAi result in lethality, it is likely that neither of them cause complete knockdown of Atg1 expression. The reason that EP3348 suppresses dPax-induced wing defects better than Atg1-RNAi is most likely due to differences in their effects on Atg1 expression. However, such differences may be subtle, and spatially restricted, as it was not possible to distinguish their effects on Atg1 expression by RTPCR of whole tissues (Chen, 2008).
It was also found that overexpression of Atg1 induces cell death and an aberrant actin cytoskeleton. When paxillin is coexpressed with Atg1, these phenotypes were enhanced, leading to increased lethality and defects in arista patterning. A deletion mutation, Df(2L)Pr.A16, which uncovers the Pax gene suppressed both Atg1- and dPax-induced defects. However, since Df(2L)Pr.A16 contains a large deletion, the possibility that the suppression effect is due to disruption of a gene that is independent of dPax cannot be ruled out. Most significantly, a marked decrease was found in the number of autophagosomes in both Pax mutant flies and paxillin-deficient mouse fibroblasts upon nutrient deprivation, thus revealing a novel requirement for paxillin in autophagy. It was also demonstrated that Atg1 can directly phosphorylate paxillin in vitro, suggesting that Atg1 may regulate the activity of paxillin. Thus far, however, it has not been possible to demonstrate that a kinase-deficient form of Atg1 detectably affects paxillin localization in mammalian cells, suggesting that the interaction of these two proteins in vivo may be more complex. Moreover, the fact that no genetic interactions were observed between other autophagy mutants and paxillin in vivo leaves open the formal possibility that the interaction between Atg1 and paxillin in Drosophila may not reflect roles for these proteins in autophagy (Chen, 2008).
Despite the identification of many autophagy-associated genes, the mechanism of autophagosome biogenesis, including the origin of membranes and how they are transported for vesicle formation, remains unclear. It has been proposed that autophagosomes form by the addition of membranes derived from ER or Golgi complex in the form of vesicle transport. Alternatively, autophagosomes could form by de novo synthesized membrane (Chen, 2008).
The precise function of Atg1 in this process is still unclear. In yeast two=hybrid screens, several proteins that directly interact with Atg1 have been identified, including Syntenin, a Rab5 GTPase.interacting protein, VAB-8, a kinesin subfamily like molecule, GABA receptor associated protein (GABARAP), and the Golgi-associated ATPase enhancer of 16kDa (GATE16). Significantly, several of these Atg1-interacting proteins are involved in membrane dynamics and vesicle trafficking, suggesting a conserved role for Atg1 in the regulation of membrane trafficking. Indeed, recent findings have implicated the mouse Atg1 ortholog, Ulk1/2, in endocytotic processes during neurogenesis (Chen, 2008).
Paxillin is a multidomain protein and functions as an essential regulatory molecule that couples integrins to the actin cytoskeleton in focal adhesions. Several paxillin-associated proteins are involved in regulation of integrin-mediated signaling associated with cell adhesion to extracellular matrix, motility and growth factor responses. The association of paxillin with Arf GAPs regulates paxillin localization, and the Arf small GTPases play a central role in membrane trafficking and cytoskeletal dynamics. While a role was found for paxillin in the regulation of membrane trafficking, whether this is related to its role in autophagy is not clear. Interestingly, the results indicate that integrin-mediated signaling is not required for paxillin's role in autophagy, suggesting that paxillin's role in the formation of autophagosomes is independent of signals from extracellular matrix. However, this aspect of the paxillin.autophagy relationship has only been examined thus far in Drosophila melanogaster, and so it remains to be determined whether the role of paxillin in autophagasome formation in mammalian cells is similarly integrin-independent (Chen, 2008).
JNK signaling functions to induce defense mechanisms that protect organisms against acute oxidative and xenobiotic insults. Using Drosophila as a model system, the role of autophagy was investigated as such a JNK-regulated protective mechanism. Oxidative stress was shown to induce autophagy in the intestinal epithelium by a mechanism that requires JNK signaling. Consistently, artificial activation of JNK in the gut gives rise to an autophagy phenotype. JNK signaling can induce the expression of several autophagy-related (ATG) genes, and the integrity of these genes is required for the stress protective function of the JNK pathway. In contrast to autophagy induced by oxidative stress, non-stress related autophagy, as it occurs for example in starving adipose or intestinal tissue, or during metamorphosis, proceeds independently of JNK signaling. Autophagy thus emerges as a multifunctional process that organisms employ in a variety of different situations using separate regulatory mechanisms (Wu, 2009).
Much interest has focused on autophagy as a mechanism by which cells defend themselves against environmental stresses. The notion that autophagy can have cell protective functions first emerged based on the finding that adaptations of several organisms to unfavorable environmental conditions require ATG genes and autophagy. Examples range from sporulation in yeast to the formation of fruiting bodies in Dictyostelium and dauer larvae in Caenorabditis elegans. Autophagy can also confer resistance to oxidative stress. Mutations that compromise the autophagy system result in increased stress sensitivity. Drosophila loss-of-function mutants for ATG7 or ATG8a, for example, are hypersensitive to H2O2 (Juhasz, 2007a; Simonsen, 2008). Cells can respond to a variety of insults, including oxidative stress, with increased autophagic activity. However, how the autophagy machinery senses and responds to stress is not thoroughly understood. Such regulation could occur at several levels, as autophagy can be regulated by transcriptional, as well as post-transcriptional mechanisms. Consistent with a function of gene regulation in this context, multiple reports show that ATG gene expression can be stimulated in response to stresses (Wu, 2009).
This work explores the control of autophagy by JNK signaling in Drosophila. The JNK pathway is an evolutionarily conserved signal transduction system that can be triggered by several types of external insults, including oxidative stress. Stress signals are conveyed by a MAP kinase cascade, which in Drosophila, consists of one of several JNKKKs (Jun kinase kinase kinases), a JNKK, the MKK7 ortholog Hemipterous (Hep) and the JNK, Basket (Bsk). The duration and extent of JNK responses is tightly controlled and restricted in time and space by a number of negative feedback mechanisms. One of these mechanisms relies on the transcriptional activation of the JNK specific MAP kinase phosphatase puckered (puc) (Wu, 2009).
JNK signaling has been implicated in the regulation of a range of cellular stress responses. These include the induction of antioxidant and repair programs, and, depending on the nature of the inducing signal and the cell type, apoptosis. Such responses can be orchestrated by changes in gene expression mediated by several transcription factors that are regulated by JNK signaling. In addition, JNK can directly regulate cell responses such as apoptosis by phosphorylating effector molecules (Wu, 2009).
It as been show that activation of the JNK pathway can protect fruit flies against oxidative toxicity. For example, flies in which JNK signaling is elevated due to the loss of one copy of the gene encoding the JNK phosphatase Puckered, or by over expressing the JNK kinase Hep, gain resistance to the free radical inducing drug paraquat (Wang, 2003; Wang, 2005; Wu, 2009).
JNK signaling emerges as a regulator of multiple mechanisms that cells can engage to increase resistance against external stresses. Recent studies in cell culture models indicate that autophagy is one of these responses. This study shows that autophagy is part of a JNK-induced stress defense program in Drosophila and explores the relationship between stress-induced autophagy and autophagy that is regulated in response to metabolic or developmental signals (Wu, 2009).
Autophagy is now recognized as a process that has multiple functions in addition to balancing energy homeostasis. It has become increasingly apparent that cells also rely on autophagy for protecting themselves against a battery of potentially harmful insults. For example, autophagy can contribute to cellular defenses against pathogens, bacterial toxins, oxidative stress and ER stress (Wu, 2009).
To explore the function and regulation in organismic stress responses, the Drosophila intestinal epithelium was studied. This tissue is directly exposed and potentially vulnerable to oxidative stress in the form of dietary toxicants or of H2O2 that can be internally generated as part of pathogen defenses. It can therefore be expected that the gut employs potent defense and regeneration systems. Exposure of the Drosophila intestine to oxidative stress, or deliberate activation of JNK signaling in the gut epithelium, results in a prominent rise of autophagosome density as monitored by Lysotracker red or GFP-LC3 staining. This effect resembles the well-established induction of autophagy in this and other tissues in response to starvation. Ultrastructure analysis by transmission electron microscopy confirms that JNK can effectively induce the formation of bona fide autophagosomes. The combined evidence from histology and microscopic analyses, the genetic interactions between JNK and ATG genes, and the induction of ATG gene expression by JNK, support the conclusion that JNK and oxidative stress can induce autophagy in the Drosophila gut (Wu, 2009).
The data suggest that JNK signaling induces autophagy, at least in part, by transcriptional activation of ATG genes. Such a mechanism would be consistent with several previous reports indicating that conditions that stimulate autophagy, such as starvation and stress, also lead to increased expression levels of ATG genes. Furthermore, the deliberate expression of ATG1, ATG6 or ATG8a by itself is sufficient to drive cells into autophagy. It is therefore plausible that the JNK-induced increases in ATG gene expression levels observed in this study might drive and/or sustain autophagy in stressed organs. However, it is also clear that autophagy can be controlled by mechanisms other than gene expression. For instance, protein phosphorylation, lipidation, and processing events have been shown to regulate the process. Two recent studies conducted in mammalian cell lines indicate that JNK can induce autophagy by phosphorylating Bcl2, thereby relieving its inhibitory effect on Beclin 1, the ATG6 homolog (Pattingre, 2008; Wei, 2008). It thus emerges that JNK may impinge on autophagy at multiple regulatory levels. Such a scenario bears an interesting resemblance of the better-understood role of JNK in the regulation of apoptosis, a process that it can control by transcriptional, as well as non-transcriptional mechanisms. It is at present a matter of speculation how these different layers of regulation are integrated and how they may have evolved. In this regard it is interesting that the only anti-death Bcl2 family member in Drosophila, the buffy gene product, does not contain JNK phosphorylation sites, suggesting that Bcl2-dependent mechanisms do not contribute to JNK induced autophagy in Drosophila. It is possible that in flies JNK acts predominantly at the transcription level, and that the role of Bcl2 in this context has evolved later (Wu, 2009).
The oxidative stress hypothesis of aging predicts that bolstering resistance against oxidative damage can extend longevity of organisms, and it has been shown previously that JNK signaling can control aging by deploying cellular oxidative stress defenses (Wang, 2003). The data presented in this study, that indicate that JNK-mediated induction of autophagy can increase oxidative stress resistance, therefore raise the question of whether such a mechanism might be also relevant for the regulation of longevity. Interestingly, several published studies support the conclusion that ATG genes, and by extension the process of autophagy, are required for the lifespan extending effects of caloric restriction or reduced Tor signaling. A recent study even finds that over expression of ATG8a under the control of one particular neuron specific promoter is sufficient to extend lifespan and confer stress resistance in Drosophila (Simonsen, 2008; Wu, 2009 and references therein).
The downstream transcription factor(s) that mediate the activation of ATG genes in response to JNK signaling are not known at this point. However, recent experiments by Juhasz indicate that the transcription factor FoxO is required for the induction of autophagy in flies that have been deprived of food (Juhasz, 2007b). In mammals it has been shown that the FoxO can induce ATG gene expression. Drosophila FoxO to be critical for JNK-mediated stress resistance. FoxO is therefore a good candidate to execute the transcriptional activation of ATG gene expression in response to JNK signaling. Further experiments are required to determine the mechanisms by which the transcriptional regulation of autophagy proceeds (Wu, 2009).
The findings presented in this study indicate that the diverse cues that can cause a cell to undergo autophagy, including metabolic, hormonal and stress signals, are transmitted by distinct signaling systems. While the JNK pathway induces autophagy in response to oxidative stress, changes in PI3K and Tor pathways stimulate autophagy in response to food deprivation and ecdysone in a JNK-independent manner. Consistent with the conclusion that the induction of autophagy in conditions of limited food supply does not involve the JNK signaling pathway, no activation of a JNK reporter gene was detected in gut cells under the starvation conditions employed in this study, which nevertheless effectively induce autophagy. However, protracted or extreme starvation may derail vital cell functions causing stress and a JNK response. For example, mammalian cells that are cultured in nutrient-deprived media activate JNK and consequently autophagy (Wu, 2009).
The induction of autophagy by oxidative stress so far discussed contrasts with developmentally programmed autophagy as it prominently occurs during metamorphosis in the Drosophila fat body. This mechanism is hormonally regulated by the ecdysone system and does not appear to require JNK (Wu, 2009).
Further genetic and molecular studies on the complex regulation of autophagy in different biological settings and organ systems are a high priority. Insight into the pleiotropic functions of this process will be valuable not only for developmental and cell biology, but also for the understanding of pathologies that are correlated with oxidative damage to cells and tissues, such as aging related and degenerative diseases (Wu, 2009).
Mutations in PINK1 and Parkin cause familial, early onset Parkinson's disease. In Drosophila, PINK1 and Parkin mutants show similar phenotypes, such as swollen and dysfunctional mitochondria, muscle degeneration, energy depletion, and dopaminergic (DA) neuron loss. PINK1 and Parkin have been shown to genetically interact with the mitochondrial fusion/fission pathway, and PINK1 and Parkin have been proposed to form a mitochondrial quality control system that involves mitophagy. However, the in vivo relationships among PINK1/Parkin function, mitochondrial fission/fusion, and autophagy remain unclear; and other cellular events critical for PINK1 pathogenesis remain to be identified. This study shows that PINK1 genetically interacte with the protein translation pathway. Enhanced translation through S6K activation significantly exacerbates PINK1 mutant phenotypes, whereas reduction of translation shows suppression. Induction of autophagy by Atg1 overexpression also rescues PINK1 mutant phenotypes, even in the presence of activated S6K. Downregulation of translation and activation of autophagy are already manifested in PINK1 mutant, suggesting that they represent compensatory cellular responses to mitochondrial dysfunction caused by PINK1 inactivation, presumably serving to conserve energy. Interestingly, the enhanced PINK1 mutant phenotype in the presence of activated S6K can be fully rescued by Parkin, apparently in an autophagy-independent manner. These results reveal complex cellular responses to PINK1 inactivation and suggest novel therapeutic strategies through manipulation of the compensatory responses (Liu, 2010; full text of article)
Previously, PINK1 and Parkin have been suggested to interact with mitochondrial fusion/fission machinery and the autophagy pathway. This study found that PINK1 also genetically interacts with the protein translation pathway. Increased global protein translation with S6K or eIF4E over expression (OE) exacerbates PINK1 mutant phenotypes, while decreased translation has the opposite effects. Overexpression of constitutively active S6Ks dramatically enhances muscle and DA neuron degeneration in PINK1 mutant flies, which can be mitigated by the co-expression of RpS6 RNAi or RpS9 RNAi, supporting that the TOR/S6K pathway modifies PINK1 mutant phenotypes through regulating global translation. Recently, it has been reported that pathogenic leucine-rich repeat kinase 2 (LRRK2), which represents the most frequent molecular lesions found in Parkinson's disease, promotes 4E-BP phosphorylation, resulting in increased eIF4E-mediated translation, enhanced sensitivity to oxidative stress, and DA neuron loss. Taken together, these results support the idea that deregulated protein translation is generally involved in the pathogenesis of Parkinson's disease (Liu, 2010).
Deregulated translation affects Parkinson's disease pathogenesis most likely at the level of energy metabolism, since protein translation is a very energy-consuming process, of which ribosomal biogenesis is the most costly, consuming approximately 80% of the energy in proliferating cells. This study shows that forced upregulation of ribosomal biogenesis in the fly muscle by the overexpression of constitutively active S6K is well tolerated in WT flies; however, such manipulation in PINK1 RNAi flies completely abolishes their flight ability, depletes ATP in the muscle and enhances muscle and DA neuron degeneration. The tolerance of increased protein translation by wild type flies is probably due to the existence of an intact mitochondrial quality control system containing PINK1 and Parkin, which can either eliminate damaged mitochondria generated during elevated energy production or minimize damages caused by increased ROS generated during energy production. However, in PINK1 or Parkin mutants that lack a functional mitochondrial quality control system, increased protein translation and the corresponding energy demand will translate into increased ROS generation, accumulation of dysfunctional mitochondria, and eventual energy depletion and tissue degeneration. Since downregulation of translation through knockdown of S6K, RpS6, or RpS9 is beneficial to PINK1 mutant flies, and S6K activity is already tuned down in PINK1 mutant flies, reduction of translation likely represents one of the cellular compensatory responses to the energy deficit caused by mitochondrial dysfunction in PINK1 mutants. Interestingly, partial reduction of S6K activity prolonges fly lifespan, whereas increased S6K activity has the opposite effects on longevity. The effects of S6K on animal lifespan and PINK1 mutant phenotypes can both be explained by the energy metabolism hypothesis and they offer a tantalizing link between aging and the pathogenesis of Parkinson's disease (Liu, 2010).
Supporting the energy metabolism model, it was shown that downregulation of protein translation by knocking down positive regulators of translation (S6K, RpS6, RpS9) or overexpressing a negative regulator (4E-BP) could rescue PINK1 mutant phenotypes. These manipulations presumably act by preserving cellular energy and reducing the workload and ROS production of mitochondria. Previously, 4E-BP OE was suggested to rescue PINK1 mutant phenotype by upregulating Cap-independent translation of stress related genes, including antioxidant genes, and boosting antioxidant gene activity has been suggested as a therapeutic strategy in the PINK1 and Parkin models of Parkinson's disease. This study found that although overexpression of antioxidant genes, such as Catalase, GTPx-1, SOD and GstS1, all showed some degree of rescue of PINK1 mutant phenotypes, their effects were in general weaker than that of Atg1 OE, Parkin OE, or Marf RNAi, particularly in the PINK1 RNAi/S6K-TE OE background. These data suggest that increasing autophagy and mitochondrial fission might be better choices to combat PINK1-related Parkinson's disease (Liu, 2010).
Autophagy is a conserved cellular process through which cytoplasmic content or defective intracellular organelles can be eliminated or recycled. Although autophagy is usually induced under adverse conditions to provide means for survival, basal level of autophagy in the cell is just as critical to the physiological health of the organism, since defects in autophagy are frequently associated with cancer, neurodegeneration, and aging. The induction of autophagy leads to the de novo formation of double membrane structure called isolation membrane, which expands to form a sealed compartment named autophagosome that will engulf materials destined for degradation. The large size of mitochondria likely poses a challenge for the autophagy machinery, as engulfment of an entire mitochondrion requires a significant amount of building materials for autophagosome formation. This is especially the case in PINK1 mutant where dysfunctional mitochondria becomes grossly swollen or aggregated. Previously, it has been shown that increased mitochondrial fission or Parkin OE could efficiently rescue the enlarged mitochondria phenotype in PINK1 mutants. The rescuing effect by increased mitochondrial fission could be due to the fact that it decreases mitochondrial size and makes it easier for the autophagosome to engulf the entire mitochondrion during mitophagy. In addition, increased mitochondrial fission could facilitate the segregation of the healthy part of a mitochondrion from the unhealthy part, thus enhancing the selective elimination of dysfunctional mitochondria through mitophagy. Supporting the mitophagy model, Parkin has been proposed to promote the efficient removal of damaged mitochondria by selectively ubiquitinating proteins on damaged mitochondria. A key prediction of the mitophagy model is that the protective effects of Parkin OE and increased mitochondrial fission as in the case of Marf RNAi will depend on the autophagy pathway. Surprisingly, this study found that blocking autophagy through Atg1 RNAi or Atg18 RNAi failed to block Parkin OE or Marf RNAi's rescuing abilities in PINK1 mutant, although Atg18 RNAi was effective in blocking the rescuing ability of Atg1 OE. This result suggests that the rescuing effect of Parkin OE or Marf RNAi is not entirely dependent on autophagy, and that other processes are likely involved. For example, Parkin has been suggested to promote mitochondrial biogenesis and regulate protein translation. Further studies are needed to elucidate the exact molecular functions of Parkin that are critically involved in mitochondrial function and tissue maintenance in vivo (Liu, 2010).
Given the well-established catabolic role of autophagy in degrading cytoplasmic contents, it helps recycle nutrients and provide energy source needed for survival under harsh conditions. In PINK1 mutants that suffer energy deficit due to mitochondrial dysfunction, induction of autophagy would present as a compensatory response to cope with the limited energy supply. Indeed, this study found that basal autophagy is induced in PINK1 mutant, and further increase of autophagy through Atg1 OE protects against PINK1 pathogenesis. Thus, decreased translation and increased autophagy both represent compensatory responses in PINK1 mutant flies, and further augmentation of these responses can effectively protect against the toxic effects of PINK1 inactivation. A previous study in cultured mammalian cells also indicated that autophagy is induced in response to PINK1 inactivation. Thus, the in vivo compensatory responses revealed in this study are likely relevant to PINK1 pathogenesis in mammals. Pharmacological interventions that promote these responses offer potential new treatment strategies for Parkinson's disease (Liu, 2010).
Autophagy is involved with the turnover of intracellular components and the management of stress responses. Genetic studies in mice have shown that suppression of neuronal autophagy can lead to the accumulation of protein aggregates and neurodegeneration. However, no study has shown that increasing autophagic gene expression can be beneficial to an aging nervous system. This study demonstrates that expression of several autophagy genes is reduced in Drosophila neural tissues as a normal part of aging. The age-dependent suppression of autophagy occurs concomitantly with the accumulation of insoluble ubiquitinated proteins (IUP), a marker of neuronal aging and degeneration. Mutations in the Atg8a gene (autophagy-related 8a) result in reduced lifespan, IUP accumulation and increased sensitivity to oxidative stress. In contrast, enhanced Atg8a expression in older fly brains extends the average adult lifespan by 56% and promotes resistance to oxidative stress and the accumulation of ubiquitinated and oxidized proteins. These data indicate that genetic or age-dependent suppression of autophagy is closely associated with the buildup of cellular damage in neurons and a reduced lifespan, while maintaining the expression of a rate-limiting autophagy gene prevents the age-dependent accumulation of damage in neurons and promotes longevity (Simonsen, 2008).
Macroautophagy (henceforth referred to as autophagy) is a highly conserved pathway that involves sequestering cytoplasmic material into double-membrane vesicles that fuse with lysosomes where the internal cargo is degraded. Autophagy occurs in response to starvation and environmental stress and has been well characterized in yeast. Recent studies in higher eukaryotes have shown that autophagy is involved in several complex cellular processes including cell death and immune response pathways. In mice, suppression of basal autophagy in the nervous system results in the accumulation of ubiquitinated proteins and neural degeneration, indicating that the continuous turnover of long-lived proteins is essential for nerve cell survival. In addition, the pathway is suppressed by insulin/ insulin-like growth factor-1 (IGF-1) signaling (through TOR kinase) and is enhanced when animals are placed on a caloric restricted diet (a well known anti-aging regime), suggesting that activation of autophagy may facilitate the removal of damaged macromolecules and organelles that accumulate during cellular aging. Protein turnover and electron microscopy studies have suggested that a functional decline in macroautophagy does occurs in older liver cells (Simonsen, 2008).
However, age-related changes in autophagy gene expression patterns have not been well studied in an organism that permits the genetic dissection of pathway function. This report addressed the role of autophagy during Drosophila aging; the overall level of autophagy gene expression is reduced by age. The age-related reduction in autophagic activity is correlated with an increased accumulation of cellular damage (build up of IUP). Further this study investigated the effect of decreased or elevated levels of Drosophila Atg8a, a member of the Atg8/LC3 protein family, on the aging fly nervous system. Atg8a mutant flies have shorter lifespans, show a dramatic accumulation of IUP and increased sensitivity to oxidative stress. In contrast, the data show that elevating the Atg8a protein in older neurons maintains the basal rates of autophagy, which is reflected in an inverse correlation with accumulation of cellular damage and a positive correlation with Drosophila longevity (increased average lifespan) (Simonsen, 2008).
The expression of select autophagy genes is downregulated in older Drosophila. To examine age-related changes in autophagy gene expression, mRNA levels of the Atg1, Atg2, Atg5, Atg8a, Atg18 and blue cheese (bchs) genes were analyzed using quantitative real-time PCR (qRT-PCR) across the entire age range of adult Drosophila lifespan and compared to message levels detected in one-day old flies. These genes represent a broad spectrum of gene function and participate at multiple stages in the pathway. The expression profiles of autophagy genes were stable (Atg1 and Atg5) or decreased significantly (Atg2, Atg8a, Atg18, bchs) by 3-weeks and remained suppressed (up to 75%) over the 9-week testing period. In contrast, the message level of the proteasome subunit rpn6 increased between 2 to 6-fold with age, in line with previous studies showing that proteasomal activity maybe upregulated with age. Together these data reveal that the expression of several essential autophagy genes decline in fly neural tissues as a normal part of aging and indicate that autophagic activity may decrease in older Drosophila (Simonsen, 2008).
Atg8a protein levels decrease in the aging CNS and in Atg8a mutant flies. To ask if there is a link between suppressed autophagy and accelerated aging, focus was placed on the Drosophila Atg8a gene, which is essential for the formation of autophagosomes and was found to have possible genetic interactions with a second autophagy protein, Bchs. The amount of Atg8a protein is also down-regulated as much as 60% by 4 weeks of age. Cytosolic Atg8 (Atg8-I) undergoes C-terminal cleavage and activation before being conjugated to lipids (Atg8-II). As a result, Atg8-II remains bound to autophagosomes throughout their formation, transport and fusion with lysosomes and has the potential to become a rate limiting component of the pathway when cellular demand for autophagy is high. Two mutant lines containing P-element insertions in the Atg8a gene (Atg8a1 or EP-UAS-Atg8a and Atg8a2) were used to examine the effects that altered gene expression has on the aging fly nervous system (Simonsen, 2008).
Atg8a1/Atg8a1 and Atg8a1/Atg8a2 mutants had reduced or absent Atg8a-I protein levels, which was confirmed by similar reductions in the Atg8a mRNA levels. The Atg8b gene is expressed at very low levels in female heads as determined by qRT-PCR, indicating that Atg8b protein level is below the detection limits of Western analysis. To determine if the age-related decline in the Atg8a message and protein could be reversed, the Drosophila Gal4/UAS system was used to drive Atg8a expression in the adult Drosophila CNS. Female flies from the APPL-Gal4 driver line (allows adult pan-neural gene expression) were crossed to males containing a UAS-P-element located in the 5' region of the Atg8a gene (EP-UAS-Atg8a, Atg8a1). While Atg8a mRNA levels were significantly reduced by age in wildtype flies, the Atg8a message remained elevated in Atg8a expressing flies for at least 4 weeks, as determined by qRT-PCR analysis. In addition, Western analysis of F1 offspring showed that the Atg8a protein declined only 20% compared to a 60% reduction in control flies. Therefore, the normal age-dependent decline seen in both the Atg8a message and protein levels in normal flies can be repressed using the APPL-Gal4 driver (Simonsen, 2008).
The accumulation of ubiquitinated proteins and aggregates in nerve cells has been observed in many human neurodegenerative diseases that are associated with aberrant protein folding and in neural tissues with suppressed autophagy. It was therefore asked whether IUP profiles change in wildtype flies as they age. Canton-S (wildtype) flies were collected at day one and at weekly intervals and their heads were processed by sequential detergent extraction. This technique allows the differential extraction of proteins based on their solubility properties in non-ionic (Triton-X) and ionic (SDS) detergents. Ubiquitinated proteins frequently accumulate in the insoluble (SDS) fraction in age-dependent neurodegenerative disorders. Western blots of SDS soluble proteins were sequentially hybridized with anti-ubiquitin and anti-actin antibodies. While young wildtype flies (day one to 3 weeks) exhibit low IUP levels, older flies (4 to 8 weeks) show a dramatic accumulation of IUP. The IUP build up is preceded by the age-dependent decrease in the expression of autophagy genes, suggesting that the progressive loss of autophagic function is a significant factor leading to compromise protein turnover by this pathway (Simonsen, 2008).
Since Atg8a levels are significantly reduced in Atg8a1/Atg8a2 mutants at week one, these flies were used to examine the effect that loss of Atg8a has on Drosophila longevity. Atg8a- (Atg8a1/Atg8a2) and control (CS) flies were, aged at 25oC and lifespan profiles determined for each genotype. Female Atg8a- flies have a 53% decrease in longevity when compared to wildtype and genotype controls. To determine whether Atg8a mutants also develop neuronal aggregates, brains of 15 day old wildtype and Atg8a- (Atg8a1/Atg8a2) flies were dissected, stained for ubiquitin and examined using confocal microscopy. Control flies had a uniform pattern of ubiquitin staining throughout the adult brain, whereas age-matched Atg8a- mutants showed formation of ubiquitinated protein inclusions in many CNS regions, including the optic lobe (OL) and subesophageal ganglia. Transmission electron microscopy analysis of brain tissue from one week-old Atg8a- flies also showed the appearance of electron dense protein aggregates or granules in the cytoplasm of neurons. These structures were primarily surrounded by a single membrane layer, but were also found without obvious membrane limitations. Microtubule-like structures could be observed that assemble with the membrane free aggregates. Similar structures are rarely seen in brains from age-matched controls. The development of protein deposits and the formation of abnormal intracellular structures are reminiscent of the CNS pathology of mice with disruption of either the Atg5 or Atg7 genes. Since suppression of autophagy is known to effect protein turnover, the IUP profiles of Atg8a mutants were examined. While young control flies (CS) had low IUP levels in SDS soluble extracts, Atg8a mutants (Atg8a1/Atg8a2 and Atg8a2) showed a significant accumulation of IUP beginning as early as one week. These data indicate that the elimination of cellular material is no longer efficient in flies with suppressed autophagy, leading to the build up of proteins and neural inclusions (Simonsen, 2008).
To assess whether enhanced Atg8a expression has an effect on the aging CNS, the lifespan profiles of F1 females and control flies maintained under standard culture conditions were examined. Elevated neuronal expression of Atg8a produces a dramatic extension of adult longevity (Simonsen, 2008).
Maximal lifespan was extended from 88 to 96 days and the average lifespan is increased 56% above that of controls. Similar results were obtained when an independent transposable construct encoding the GFP-Atg8a protein is expressed in the brains of both male and female flies. Lifespan extension was not seen when Atg8a was expressed using an early pan-neural driver line. Expression of two other autophagy genes (Atg2 and bchs) or other proteins associated with enhanced longevity (Hsc70 and GST) using the APPL-Gal4 driver did not extend adult Drosophila lifespan to the same extent as the Atg8a protein. The difference between the APPL-Gal4 and ELAV-Gal4 expression of Atg8a is likely related to the age-dependent expression differences of each Gal4-driver, suggesting that the timing of Atg8a expression in the aging CNS is critical for its ability to enhance longevity. Elevated Atg8a expression is also protective when flies are maintained at higher temperatures (29oC), under conditions known to accelerate Drosophila aging. Since wild type Drosophila have a dramatic increase in IUP profiles starting at 4 weeks and Atg8a mutants show accelerated IUP accumulation, it was asked whether increased neuronal expression of Atg8a could prevent the buildup of IUP that naturally occurs with age. Control flies (CS), Atg8a1/Atg8a1 (Atg8a-) and Atg8a expressing flies (Atg8a+) were aged for 4 weeks and IUP levels from SDS head extracts were examined by Western analysis. Control (CS) and Atg8a- fliesshowed a significant accumulation of IUP that is typical for both genotypes at this age. In contrast, age-matched Atg8a+ animals showed a 12-fold reduction in IUP levels. These data clearly show that the decrease in autophagy normally occurring with age correlates with IUP accumulation and suggests that elevated levels of a rate-limiting component of autophagy can facilitate the clearance of ubiquitinated or aggregate-prone proteins later in life (Simonsen, 2008).
As a consequence of a normal aerobic metabolism cells are exposed to reactive oxygen species (ROS), which can cause direct damage to macromolecules. There is also an increase in oxidative damage associated with age and age-related neurodegenerative diseases. To determine if autophagy affects the acute oxidative stress response in the Drosophila nervous system, control, Atg8a1/Atg8a2 mutant or Atg8a expressing (APPL-Gal4/EP-UAS-Atg8a) flies were placed on to media containing 1.5% H2O2 and analyzed their lifespan profiles. While suppression of autophagy resulted in a shortened lifespan, Atg8a expressing flies exhibited longer lifespans than controls in the presence of oxidants. One potential mechanism for autophagy to regulate macromolecular damage caused by oxidant exposure involves the direct removal of ROS damaged proteins. Previous studies have measured damage by examining the accumulation of IUP or carbonylated protein levels in neural tissues. Therefore, both parameters were examined after exposing duplicate sets of control, Atg8a mutant and Atg8a expressing female flies to normal media (-) or media containing 1.5% H2O2 (+) for 24 hours. IUP levels increased on average 20% following H2O2 exposure in control flies. Atg8a mutants show a dramatic 126% increase in IUP, whereas flies with elevated neuronal Atg8a have a marked reduction in IUP accumulation relative to control flies. In a parallel study, control and Atg8a mutant flies showed a pronounced accumulation of several carbonylated proteins. In contrast, upregulating Atg8a dramatically lowers the level of damaged proteins following H2O2 treatment. Taken together, these data indicate that autophagic activity is inversely correlated with lifespan and accumulation of ROS-modified proteins following exposure to oxidative stress (Simonsen, 2008).
This study has demonstrate for the first time that maintaining the bulk clearance pathway of macroautophagy in a mature nervous system promotes longevity and reduces markers of cellular aging like IUP. This work also demonstrates that several key pathway members are suppressed at the level of gene transcription as a normal part of Drosophila aging. The age-dependent decrease in autophagy gene expression is paralleled by a pronounced accumulation of IUP (Simonsen, 2008).
Consistent with the hypothesis that the progressive loss of autophagic function results in the accumulation of aging markers, Atg8a mutant flies also have a reduced lifespan, increased sensitivity to oxidative stress and morphological phenotypes consistent with premature or accelerated aging. Both mutational loss and an age-dependent decline in autophagy decreases the pathway's ability to serve as the bulk clearance mechanism for cellular damage, which can go on to further impair the long-term function of neurons. The loss-of-function phenotypes seen in mutant Drosophila have striking similarities to those characterized in some of the most common human neurodegenerative disorders associated with misfolded protein, and in mouse models in which basal autophagy is suppressed in the brain. This diverse data underscores the functional conservation of the pathway and suggests that the age-dependent suppression of autophagy may be a contributing factor for human disorders (Simonsen, 2008).
Insulin/IGF-1 signaling and caloric restriction have been shown to be major determinants of aging. Most studies examining the link between aging and Insulin/IGF-1/CR-mediated signaling have focused on downstream mediators such as the forkhead transcription factors and sirtuins. However, a recent study in C. elegans revealed that the enhanced longevity phenotype of an insulin-signaling mutant is negated by decreased expression of the beclin-1/Atg6 gene, suggesting that caloric restriction and the insulin/TOR signaling may also affect lifespan via autophagic pathways. This study has demonstrated that circumventing upstream signaling pathways and directly maintaining the expression of an essential autophagy gene (At8ga) in the aging nervous system leads to a dramatic extension of lifespan and resistance to oxidative stress. This information and the placement and function of Atg8/LC3 within the pathway and its degradation by the lysosome suggest it may become a rate-limiting by directly enhancing Atg8a expression. These results suggest that upregulation and the supplementation of rate-limiting components of the autophagic pathway may also be beneficial for the health and maintenance of the human nervous system under a wide variety of stressful conditions that involve oxidant exposure, misfolded proteins and simply old age (Simonsen, 2008).
Autophagy is a catabolic process that is negatively regulated by growth and has been implicated in cell death. This study finds that autophagy is induced following growth arrest, and precedes developmental autophagic cell death of Drosophila salivary glands. Maintaining growth by expression of either activated Ras or positive regulators of the class I phosphoinositide 3-kinase (PI3K) pathway inhibits autophagy and blocks salivary gland cell degradation. Developmental degradation of salivary glands is also inhibited in autophagy gene (atg) mutants. Caspases are active in PI3K-expressing and atg mutant salivary glands, and combined inhibition of both autophagy and caspases increases suppression of gland degradation. Further, induction of autophagy is sufficient to induce premature cell death in a caspase-independent manner. These results provide in vivo evidence that growth arrest, autophagy, and atg genes are required for physiological autophagic cell death, and that multiple degradation pathways cooperate in the efficient clearance of cells during development (Berry, 2007).
These studies indicate that arrest of PI3K-dependent growth is an important determinant of autophagic cell death of salivary glands during Drosophila development. Maintenance of growth by expression of either activated Ras, Dp110, or Akt in salivary glands is sufficient to inhibit salivary gland degradation. It is possible that the larger Dp110-, Akt- and RasV12-expressing salivary glands simply have more material to degrade, and this is why they persist. It is suspected that this is not the case, however, since Dp110-expressing glands are larger than RasV12-expressing glands, yet RasV12-expressing glands are less degraded. Although PI3K-dependent growth inhibits autophagy, growth could influence other downstream targets. However, the Atg1-induced suppression of the Dp110 persistent salivary gland phenotype and the persistence of vacuolated salivary gland cell fragments in atg loss-of-function mutants support the conclusion that growth arrest and autophagy are required for proper salivary gland degradation (Berry, 2007).
Ras and class I PI3K signaling are complex, and cross-talk occurs between these pathways. Although the data indicate that both activated Ras and PI3K have similar effects on salivary gland cell growth and inhibition of autophagy, it is observed that Ras-expressing cells were more intact 24 hours apf. Caspase activity is detected in Dp110-and Akt-expressing glands, and it is speculated that part of the degradation observed in Dp110 and Akt glands was due to caspases. Indeed, combining Dp110 expression with caspase inhibition resulted in intact salivary glands. This additive phenotype indicates that multiple degradation pathways are involved in autophagic cell death in vivo. Caspases were also active in Ras-expressing glands that were predominantly intact; thus activated Ras likely influences factors separate from caspases and the PI3K pathway. Ras regulates PI3K-independent pathways including MAPK and the cell cycle. Proliferating cells usually double in size prior to division, and because of this, cell growth and division are often considered synonymous. These studies demonstrate that although expression of either Myc or CyclinD with Cdk4 is sufficient to induce nuclear size, they do not inhibit salivary gland degradation. These data support the conclusion that growth arrest, but not cell cycle arrest, is an important determinant of salivary gland autophagic cell death. While many studies have defined relationships between cell cycle arrest and cell death, this study defines a unique relationship between cell growth arrest and cell death (Berry, 2007).
Given autophagy’s well established function in cell survival, a role for autophagy in cell death seems paradoxical. The discovery that caspases function in cells dying with a Type II autophagic morphology led to speculation that all programmed cell death is regulated by apoptosis factors. Further, the preponderance of in vitro evidence shows a role for autophagy in cell death when caspases or apoptosis factors are inhibited. This study found that reduced function of any one of seven atg genes inhibits salivary gland degradation. The incomplete degradation of salivary glands in multiple atg loss-of-function mutants provides the first in vivo evidence that autophagy and atg genes are required for proper degradation of cells during developmental cell death. Caspase activity and caspase-dependent DNA fragmentation occurs in these atg mutants, indicating that autophagy is a caspase-independent degradation pathway required for complete cell degradation in autophagic cell death during development. Further, induction of autophagy by Atg1 expression leads to premature caspase-independent salivary gland degradation. The data do not exclude a role for caspases in autophagic cell death. Either inhibition of caspases by p35 or reduced atg gene function result in delayed and incomplete degradation of salivary glands, and the combined inhibition of caspases with reduced atg function results in increased persistence of this tissue. These data suggest that autophagy and caspases function in parallel pathways during salivary gland cell death, and that both independently contribute to cell destruction. Further, the presence of both autophagy and caspases appears to be more typical of autophagic cell death that occurs under physiological conditions. Autophagic cell death models of mammary lumen formation and embryonic cavitation, as well as amphibian developmental cell death, all involve both processed caspase-3 and autophagy (Berry, 2007).
The designations of type I apoptotic death and type II autophagic death are based on morphological criteria. The current studies indicate that cell morphology likely reflects difference in the factors that are used to activate cell death and degrade the dying cell. The degradation of salivary glands in caspase mutants indicates that caspase-independent factors are involved in autophagic cell death. The presence of autophagosomes in dying salivary glands led to an investigation of cell death in atg gene mutants; stronger defects were observed in salivary gland degradation with perturbed atg gene function than with drice, ark, and dronc mutants. These data indicate that cell morphology is informative, given that it suggested autophagy is involved in the death of salivary glands. However, it is important to note that cell death classification that is based on morphology can be misleading, since salivary glands clearly use both caspases and autophagy, degradation mechanisms that had been speculated to be strictly associated with a single morphological form of cell death (Berry, 2007).
Now that it is clear that autophagy participates in cell death under some circumstances, it will be critical to determine how autophagy participates in cell killing and removal. A recent study showed that autophagy is required to generate the energy needed to promote phagocytosis signaling in an in vitro model of embryonic cavitation (Qu, 2007). This is not believed to be the same as the role of autophagy in salivary glands, since no phagocytosis is observed during salivary gland death. Alternatively, autophagy may be used to recruit and degrade factors that promote cell survival, such as the degradation of cytoplasmic catalase in mouse L929 cells. Finally, extreme levels of autophagy may be sufficient to cause a metabolic catastrophe by degrading substrates and mitochondria that are needed for energy. The latter possibility does exist in salivary glands, as expression of the Atg1 kinase is sufficient to induce the death of fat (Scott, 2007) and salivary gland cells. Unlike fat cells, elevated autophagy does not induce caspase-dependent DNA fragmentation in salivary gland cells, and expression of p35 does not inhibit Atg1-induced death (Berry, 2007).
The prevalence of apoptosis and the potent killing potential of caspases raise the question of why autophagy participates in developmental cell death. In the context of Drosophila and other insects, larval cells have a modified endoreplication cell cycle that results in the production of gigantic cells. The number and size of cells may prohibit engulfment and digestion by phagocytes, and autophagy may be necessary for self-degradation. Further, the life history of the organism may informative as to why autophagy participates in the destruction of tissues. Drosophila do not feed during the 3 day period of metamorphosis. Thus, the differentiation and morphogenesis of the entire adult occurs in the absence of food, and the resources to build the adult fly must come from reserves that are set aside during larval development. One important source of these resources is the fat that exhibits elevated levels of autophagy at the onset of metamorphosis. Several other large larval tissues are destroyed by autophagic cell death during metamorphosis including the midgut and salivary glands. It is speculated that like fat, catabolism of these tissues by autophagy provides resources that are needed to construct the adult. Similarly, it is speculated that the large number of autophagosomes observed in dying amphibian cells may serve to recycle nutrients during metamorphosis when these animals do not feed (Berry, 2007).
These studies have indicated that it is necessary to be cautious when considering autophagy to be either a cell survival or cell death process. Perhaps it is useful to consider autophagy for what it is; a catabolic process that contributes to many cellular and biological processes. This is not that different from the caspase proteases that are widely considered to be apoptosis proteases, as it is now clear that caspases also function in cell differentiation. Future studies are likely to show that autophagy functions in many cell types, and that its contribution to cell survival and cell death are dependent on the type and physiological context of the cell (Berry, 2007).
It has been proposed that cell growth and autophagy are coordinated in response to cellular nutrient status, but the relationship between them is not fully understood. This study characterized the fly mutants of Autophagy-specific gene 1 (ATG1), an autophagy-regulating kinase, and it was found that ATG1 is a negative regulator of the target of rapamycin (TOR)/S6 kinase (S6K) pathway. The Drosophila studies have shown that ATG1 inhibits TOR/S6K-dependent cell growth and development by interfering with S6K activation. Consistently, overexpression of ATG1 in mammalian cells also markedly inhibits S6K in a kinase activity-dependent manner, and short interfering RNA-mediated knockdown of ATG1 induces ectopic activation of S6K and S6 phosphorylation. Moreover, ATG1 specifically inhibits S6K activity by blocking phosphorylation of S6K at Thr 389. Taken together, these genetic and biochemical results strongly indicate crosstalk between autophagy and cell growth regulation (Lee, 2007).
Previous studies have demonstrated that homozygous EP3348 flies, in which a single P element is inserted into the 5' untranslated region of DmATG1, show larval/pupal lethality. However, after the genomic background of the EP3348 line was cleared by four backcrosses with w1118 flies, about 30% of homozygous mutants were found to develop to adults. To confirm that the P-element insertion in EP3348 hampers transcription of DmATG1 (Scott, 2004), quantitative real-time reverse transcriptase-PCR (qRT-PCR), was performed. This showed highly reduced DmATG1 expression in the mutant. Therefore, this cleared EP3348 allele was named DmATG11 (Lee, 2007).
The mitotic phenotypes in the larval brain and imaginal discs of DmATG1 mutants, DmATG11 and DmATG1Δ3d (a null allele of DmATG1 were examined. However, no notable mitotic defects could be found in the mutants compared with the wild-type control (w1118. Consistently, DmATG1 mutants had no gross chromosomal abnormalities in the neuroblast cells of third instar larval brains (Lee, 2007).
Next, defects in autophagy in DmATG11 flies were examined using toluidine blue-azure II staining and transmission electron microscopy (TEM) analyses. As expected, DmATG11 flies showed marked defects in the induction of autophagy under conditions of starvation. This is highly consistent with the previous study using DmATG1Δ3d (Scott, 2004). These results strongly supported that DmATG11 is a new hypomorphic mutant allele of DmATG1 (Lee, 2007).
To understand further the in vivo roles of ATG1 in Drosophila, double-mutant lines were generated for dTORP1 (a loss-of-function mutant allele of dTOR and DmATG1 mutants (DmATG11, DmATG1Δ3d and EP3348). Homozygous dTORP1 mutants showed growth arrest in the second/early third instar larval stage and markedly delayed growth. Surprisingly, homozygous dTORP1 larvae with a heterozygous genetic background of DmATG11 or DmATG1Δ3d not only grew faster than homozygous dTORP1, but also extended their developmental stage to the mid-late third instar larval stage. Furthermore, the double-homozygous mutants between dTORP1 and various DmATG1 mutants survived up to the mid-late third instar larval stage, which is inconsistent with the previous results (Scott, 2004; Lee, 2007).
In addition, it was found that another phenotype of dTORP1 mutants, lipid vesicle aggregation in the fat body, was also suppressed by a reduction of the gene dosage of DmATG1. These results implicated that ATG1 negatively mediates the developmental and physiological roles of TOR in Drosophila (Lee, 2007).
Since dTOR regulates cell growth in a cell-autonomous manner, whether DmATG1 is also involved in this role of dTOR was examined. The cell and nuclear sizes of the salivary gland cells were markedly reduced in dTORP1 larvae. Intriguingly, the heterozygous genetic background of DmATG11 or DmATG1Δ3d partly rescued the reduced cell and nuclear size phenotype of dTORP1, strongly implicating that DmATG1 mediates the crucial function of dTOR in cell growth regulation. This is further supported by recent results, that DmATG1-null cells have a relative growth advantage over wild-type cells when treated with rapamycin, a specific inhibitor of dTOR (Lee, 2007).
To examine the possibility that the suppression of dTORP1 phenotypes by DmATG1 mutation resulted from altered auto-phagic activities, the genetic interactions were investigated between dTOR and other autophagy-related genes, such as ATG6 (Beclin1) and UVRAG (UV radiation resistance associated gene), which are known to form a complex regulating autophagosome formation (Liang, 2006). As a result, ATG6 and UVRAG mutations did not suppress the developmental delay and cell growth defects of dTOR mutants, showing that the interaction between dTOR and DmATG1 is not caused indirectly by uncontrolled regulation of autophagy (Lee, 2007).
To determine the functional interaction between ATG1 and S6K, a downstream effector of TOR, double-mutant lines were generated between DmATG11 and dS6K mutants -- a hypomorphic allele, dS6K07084, and a null allele, dS6Kl−1. Using TEM analyses, it was observed that a reduction of dS6K gene dosage did not rescue the defects in autophagosome formation in starved DmATG11 homozygous larvae. However, surprisingly, the reduced gene dosage of dS6K increased the eclosion rate of homozygous DmATG11 in a dS6K gene dosage-dependent manner. Although the possibility that S6K promotes autophagy as reported previously (Scott, 2004) cannot be excluded, these data indicate that dS6K has an important role in DmATG1-dependent developmental processes (Lee, 2007).
Next, a biochemical analysis was conducted to examine the effect of loss of DmATG1 on dS6K activation. As a result, it was found that dS6K was markedly activated (~threefold increase) in homozygous DmATG11 larvae and pupae, measured by the phosphorylation of dS6K at the Thr 398 site, compared with the wild-type controls. Notably, the increased level of dS6K phosphorylation in DmATG11 mutants was about one-third of that in flies overexpressing Rheb. Consistent with this, DmATG1 overexpression almost completely inhibited dS6K Thr 398 phosphorylation in Drosophila (Scott, 2007). These results strongly suggest an important role of ATG1 in the regulation of S6K (Lee, 2007).
To extend these findings to the mammalian system and also to investigate further the molecular mechanism of the interaction between ATG1 and TOR/S6K, the effect of ATG1 on the activity of S6K was examined in mammalian cells. There are two isoforms of ATG1 in mammals, which are named UNC-51-like kinase (ULK) 1 and 2 (Zhou, 2007). However, according to the agreement on gene nomenclature made by researchers in the field of autophagy, they were renamed ATG1α and ATG1β, respectively. Nutrient deprivation of HEK293T cells abolished the phosphorylation of S6K at both Thr 229 and Thr 389 sites, which represents the activation status of S6K . When nutrients including amino acids and glucose (DMEM) were added back to the cells, the phosphorylation of both sites in S6K was strongly induced. However, co-expression of wild-type mouse ATG1α (ATG1α WT) strongly inhibited S6K activity induced by DMEM. On the contrary, a kinase-dead form of ATG1α (ATG1α KI) was not able to block the nutrient-induced activation of S6K, showing that ATG1α inhibits S6K in a kinase activity-dependent manner. Consistently, epidermal growth factor (EGF)-stimulated S6K activation was also inhibited by ATG1α. Furthermore, ATG1β, another isoform of ATG1, has the same inhibitory effect on S6K phosphorylation as ATG1α. These data strongly suggest that ATG1 regulates the activities of upstream kinases or phosphatases of S6K, which affect both Thr 229 and Thr 389 phosphorylation (Lee, 2007).
As ATG1 is a crucial regulator of autophagy in yeast and Drosophila, whether overexpression of ATG1 can induce autophagy in mammalian cells was tested. Nutrient deprivation was able to induce autophagy in MCF-7 cells, whereas overexpression of ATG1 did not induce autophagy in MCF-7 and HEK 293T cells, indicating that the inhibition of TOR/S6K by ATG1 is not an indirect consequence of an ectopic induction of autophagy. This was further supported by the observation that overexpression of ATG6 and UVRAG did not inhibit the phosphorylation of S6K, which is also highly consistent with the above Drosophila data (Lee, 2007).
Then, short interfering RNA (siRNA) targeting was used for ATG1α and ATG1β messenger RNA to confirm that ATG1 inhibits S6K activity. The efficacy of siRNA was verified by qRT–PCR using ATG1-specific primers. Transfection of ATG1α and ATG1β siRNA to HEK 293T cells led to increased phosphorylation of S6K Thr 389 and S6 (the only proven in vivo substrate of S6K) Thr 235/236. This result was further supported by immunocytochemistry by using phosphospecific S6 antibody; ATG1 siRNA transfection alone induced phosphospecific immunostaining of S6 in starved cells. Taken together, these results clearly demonstrate that ATG1 inhibits S6K and S6 in vivo (Lee, 2007).
Notably, the level of S6K activation by ATG1 siRNA was about 5% of that by nutritional stimulation. This weak activation of S6K resulted from partial gene knockdown by RNAi. Consistent with this conclusion, more pronounced activation of dS6K was observed in DmATG1 mutants in Drosophila, which contains only a single orthologue of ATG1 (Lee, 2007).
S6K is in the AGC kinase family, which also includes Akt and p90 ribosomal S6 kinase (RSK). These kinases are regulated by a similar mechanism in which both phosphorylation at their activation loop and a hydrophobic motif next to the kinase domain are required for full activation. 3-Phosphoinositide-dependent kinase 1 (PDK1) is a kinase responsible for phosphorylation at the activation loop of AGC kinases. In the case of S6K, PDK1 directly phosphorylates the Thr 229 residue at the activation loop of S6K, which is strictly dependent on the previous phosphorylation of Thr 389 at the hydrophobic motif. These motifs are well conserved among the family members in different species. Therefore, whether ATG1α also affects the phosphorylation of Akt and RSK was examined. Interestingly, the phosphorylation of Akt and RSK was not affected by ATG1α, with or without stimulation by insulin and EGF. These data indicate that ATG1α specifically modulates S6K activity (Lee, 2007).
Next, to understand the molecular mechanism of the specific regulation of S6K by ATG1, whether ATG1 affects the phosphorylation of Thr 229 in S6K was investigated by using an S6K mutant that specifically mimics the phosphorylated form of S6K, Thr 389 Glu. As a result, Thr 229 phosphorylation of the S6K Thr 389 Glu mutant was not affected by wild-type ATG1. Because Akt was not inhibited by ATG1, it is unlikely that ATG1 regulates Thr 389 phosphorylation of S6K by inhibiting the PDK1/Akt signalling module. Therefore, it is believed that ATG1 modulates S6K activity by affecting S6K Thr 389-specific kinases or phosphatases (Lee, 2007).
In summary, under nutrient-rich conditions, activation of TOR leads to inhibition of ATG1, which facilitates S6K Thr 389 phosphorylation and the subsequent phosphorylation of Thr 229 by PDK1 to activate S6K fully. Consequently, activated S6K promotes cell growth. However, under conditions of starvation, TOR becomes quiescent and ATG1 can inhibit S6K by blocking Thr 389 phosphorylation. This nutrient-dependent signalling switch operated by TOR and ATG1 is highly consistent with that in yeast. The observations described in this study clearly show the presence of crosstalk between ATG1 and S6K signalling, in which ATG1 specifically inhibits S6K. This study also showed that this is evolutionarily conserved between Drosophila and mammals. It is believed that these biochemical data and the fly system will be useful in future studies that address the detailed molecular mechanism of crosstalk between the two nutrition-dependent physiological processes -- autophagy and cell growth (Lee, 2007).
In response to starvation, eukaryotic cells recover nutrients through autophagy, a lysosomal-mediated process of cytoplasmic degradation. Autophagy is known to be inhibited by TOR signaling (see Drosophila Tor), but the mechanisms of autophagy regulation and its role in TOR-mediated cell growth are unclear. Signaling through TOR and its upstream regulators PI3K and Rheb is necessary and sufficient to suppress starvation-induced autophagy in the Drosophila fat body. In contrast, TOR's downstream effector S6K promotes rather than suppresses autophagy, suggesting S6K downregulation may limit autophagy during extended starvation. Despite the catabolic potential of autophagy, disruption of conserved components of the autophagic machinery, including ATG1 and ATG5, does not restore growth to TOR mutant cells. Instead, inhibition of autophagy enhances TOR mutant phenotypes, including reduced cell size, growth rate, and survival. Thus, in cells lacking TOR, autophagy plays a protective role that is dominant over its potential role as a growth suppressor (Scott, 2004).
Autophagy likely evolved in single-cell eukaryotes to provide an energy and nutrient source allowing temporary survival of starvation. In yeast, Tor1 and Tor2 act as direct links between nutrient conditions and cell metabolism. These proteins sense nutritional status by an unknown mechanism, and effect a variety of starvation responses including changes in transcriptional and translational programs, nutrient import, protein and mRNA stability, cell cycle arrest, and induction of autophagy. Autophagy thus occurs in the context of a comprehensive reorganization of cellular activities aimed at surviving low nutrient levels (Scott, 2004).
In multicellular organisms, TOR is thought to have retained its role as a nutrient sensor but has also adopted new functions in regulating and responding to growth factor signaling pathways and developmental programs. Thus in a variety of signaling, developmental, and disease contexts, TOR activity can be regulated independently of nutritional conditions. In these cases, autophagy may be induced in response to downregulation of TOR despite the presence of abundant nutrients and may potentially play an important role in suppressing cell growth rather than promoting survival. Identification of the tumor suppressors PTEN, and TSC1 and TSC2 as positive regulators of autophagy provides correlative evidence supporting such a role for autophagy in growth control. Alternatively, since TOR activity is required for proper expression and localization of a number of nutrient transporters, inactivation of TOR may lead to reduced intracellular nutrient levels, and autophagy may therefore be required under these conditions to provide the nutrients and energy necessary for normal cell metabolism and survival (Scott, 2004).
The results presented here provide genetic evidence that under conditions of low TOR signaling, autophagy functions primarily to promote normal cell function and survival, rather than to suppress cell growth. This conclusion is based on the finding that genetic disruption of autophagy does not restore growth to cells lacking TOR, but instead exacerbates multiple TOR mutant phenotypes. It is important to note that mutations in TOR do not disrupt larval feeding, and thus disruption of autophagy is detrimental in TOR mutants despite the presence of ample extracellular nutrients. The finding that autophagy is critical in cells lacking TOR further supports earlier studies suggesting that inactivation of TOR causes defects in nutrient import, resulting in an intracellular state of pseudo-starvation (Scott, 2004).
Can the further reduction in growth of TOR mutant cells upon disruption of autophagy be reconciled with the potential catabolic effects of autophagy? TOR regulates the bidirectional flow of nutrients between protein synthesis and degradation through effects on nutrient import, autophagy, and ribosome biogenesis. When TOR is inactivated, rates of nutrient import and protein synthesis decrease, resulting in a commensurate reduction in mass accumulation and cell growth. In addition, autophagy is induced to maintain intracellular nutrient and energy levels sufficient for normal cell metabolism. When autophagy is experimentally inhibited in cells lacking TOR, this reserve source of nutrients is blocked, leading to a further decrease in energy levels, protein synthesis, and growth. It is noted that autophagy may have additional functions in cells with depressed TOR signaling, including recycling of organelles damaged by the absence of TOR activity, or selective degradation of cell growth regulators, analogous to the regulatory roles of ubiquitin-mediated degradation (Scott, 2004).
Autophagy is required for normal developmental responses to inactivation of insulin/PI3K signaling in the nematode C. elegans. In response to starvation or disruption of insulin/PI3K signaling, C. elegans larvae enter a dormant state called the dauer. Autophagy has been observed in C. elegans larvae undergoing dauer formation: disruption of a number of ATG homologs interfers with normal dauer morphogenesis. Importantly, simultaneous disruption of insulin/PI3K signaling and autophagy genes results in lethality, similar to the results presented in this study. Thus despite significant differences in developmental strategies for surviving nutrient deprivation, autophagy plays an essential role in the starvation responses of yeast, flies, and worms (Scott, 2004).
The prevailing view that S6K acts to suppress autophagy was founded on correlations between induction of autophagy and dephosphorylation of rpS6 in response to amino acid deprivation or rapamycin treatment. However, the genetic data presented in this study argue strongly against a role for S6K in suppressing autophagy: unlike other positive components of the TOR pathway, null mutations in S6K do not induce autophagy in fed animals. It is suggested that the observed correlation between S6K activity and suppression of autophagy is due to common but independent regulation of S6K and autophagy by TOR. Thus, autophagy suppression and S6K-dependent functions such as ribosome biogenesis represent distinct outputs of TOR signaling (Scott, 2004).
How might TOR signal to the autophagic machinery, if not through S6K? In yeast, this is accomplished in part through regulation of Atg1 kinase activity and ATG8 gene expression (Kamada, 2000 and Kirisako, 1999). The demonstration of a role for Drosophila ATG1 and ATG8 homologs [see TG8a (CG32672) and ATG8b (CG12334)] in starvation-induced autophagy, and the genetic interaction observed between ATG1 and TOR, are consistent with a related mode of regulation in higher eukaryotes. However, it is noted that other components of the yeast Atg1 complex such as Atg17 and Atg13, whose phosphorylation state is rapamycin sensitive, do not have clear homologs in metazoans, indicating that differences in regulation of autophagy by TOR are likely (Scott, 2004).
In addition to excluding a role for S6K in suppression of autophagy, these results reveal a positive role for S6K in induction of autophagy. S6K may promote autophagy directly, through activation of the autophagy machinery, or indirectly through its effects on protein synthesis. The latter possibility is consistent with previous reports that protein synthesis is required for expansion and maturation of autophagosomes. Interestingly, despite being required for autophagy, S6K is downregulated under conditions that induce it, including chronic starvation and TOR inactivation. Consistent with this, it was found that lysotracker staining is significantly weaker in chronically starved animals or in TOR mutants than in wild-type animals starved 3-4 hr. Furthermore, expression of constitutively activated S6K has no effect in wild-type, but restores lysotracker staining in TOR mutants to levels similar to those of acutely starved wild-type animals. It is suggested that downregulation of S6K may limit rates of autophagy under conditions of extended starvation or TOR inactivation and that this may protect cells from the potentially damaging effects of unrestrained autophagy (Scott, 2004).
Co-culture and conditioned media experiments have shown that the Drosophila fat body is a source of diffusible mitogens. The fat body has also been shown to act as a nutrient sensor through a TOR-dependent mechanism and to regulate organismal growth through effects on insulin/PI3K signaling. The results in this study extend these findings by showing that this endocrine response is accompanied by the regulated release of nutrients through autophagic degradation of fat body cytoplasm. Preventing this reallocation of resources, either through constitutive activation of PI3K or through inactivation of ATG genes, results in profound nutrient sensitivity. Thus, in response to nutrient limitation, the fat body is capable of simultaneously restricting growth of peripheral tissues through downregulation of insulin/PI3K signaling and providing these tissues with a buffering source of nutrients necessary for survival through autophagy (Scott, 2004).
Axonal transport mediated by microtubule-dependent motors is vital for neuronal function and viability. Selective sets of cargoes, including macromolecules and organelles, are transported long range along axons to specific destinations. Despite intensive studies focusing on the motor machinery, the regulatory mechanisms that control motor-cargo assembly are not well understood. This study shows that UNC-51/ATG1 kinase regulates the interaction between synaptic vesicles and motor complexes during transport in Drosophila. UNC-51 binds UNC-76, a kinesin heavy chain (KHC) adaptor protein. Loss of unc-51 or unc-76 leads to severe axonal transport defects in which synaptic vesicles are segregated from the motor complexes and accumulate along axons. Genetic studies show that unc-51 and unc-76 functionally interact in vivo to regulate axonal transport. UNC-51 phosphorylates UNC-76 on Ser(143), and the phosphorylated UNC-76 binds Synaptotagmin-1, a synaptic vesicle protein, suggesting that motor-cargo interactions are regulated in a phosphorylation-dependent manner. In addition, defective axonal transport in unc-76 mutants is rescued by a phospho-mimetic UNC-76, but not a phospho-defective UNC-76, demonstrating the essential role of UNC-76 Ser(143) phosphorylation in axonal transport. Thus, these data provide insight into axonal transport regulation that depends on the phosphorylation of adaptor proteins (Toda, 2008).
This study demonstrates that loss of unc-51 function affects the transport of several axonal cargoes, including SV and mitochondria. In unc-51 mutants, SV transport is severely attenuated and many SV aggregates are found along the larval SGN axons. SV aggregation in unc-51 mutants is similar to that observed in Khc mutants. However, unlike Khc mutants, SV aggregates in unc-51 mutants do not contain mitochondria, suggesting that the aggregated SVs in unc-51 mutants do not cause overall 'steric hindrance,' in which the impaired cargo interferes with the transport of other cargoes as a secondary effect. In unc-51 mutants, Rab5-positive membranes also exhibited a pattern of aggregation different from that for SVs, supporting the idea that loss of unc-51 results in defective axonal transport in a membrane type-dependent manner. This view is further supported by a recent analysis of unc-51 mutants in C. elegans that showed that only a subset of cargoes are selectively mislocalized, whereas a majority of other cargoes are not affected (Toda, 2008).
SVs are one of the most severely affected axonal cargoes in unc-51 mutants. Two anterograde motors, kinesin-1 and kinesin-3, have been implicated in SV transport in Drosophila. A recent study addressed an essential role of unc-104 (imac, kinesin-3) in SV transport (Pack-Chung, 2007). Virtually all SVs fail to enter axons and accumulate in neuronal cell bodies during the embryonic period. In contrast, mutations in Khc, a catalytic component of kinesin-1, and also mutations in Klc, an accessory component of kinesin-1, cause SV accumulations within axons of larval SGNs. These studies suggest that, although kinesin-3 is primarily responsible for SV transport, kinesin-1 plays a role in SV transport in a manner distinct from that for kinesin-3, or the two motors may have complementary roles in SV transport at the larval stage. The unc-51 mutant phenotypes, as well as the biochemical evidence that UNC-51 forms a complex with UNC-76/KHC, are most consistent with the idea that UNC-51 functions in SV transport through a kinesin-1-dependent pathway (Toda, 2008).
In unc-51 mutants, SVs accumulate within axons at sites distant from cell bodies, suggesting that SVs could partially transport along axons in the absence of unc-51. It is possible that maternally deposited unc-51 contributes to partial transport of SVs into axons, as suggested for defective SV transport in Khc mutants. It is also possible that there are specific subcellular locations (e.g., axon hillock) or earlier developmental periods where SV transport does not require unc-51 activity. The axonal SV accumulation in unc-51 mutants could be a result of spatially distinct requirement of unc-51 activity for maintaining SV-motor integrity during transport. In addition, kinesin-3 likely contributes to SV transport in unc-51 mutants, resulting in translocation of a subset of SVs into axons and to synapses. SVs that are localized to unc-51 mutant NMJs may reflect a subpopulation of those that were carried by kinesin-3 and did not need unc-51 activity for their transport. In summary, the cooperative action of multiple pathways, including unc-51, unc-76, kinesin-1 and kinesin-3, may be necessary for complete SV transport (Toda, 2008).
Previous in vitro studies that addressed a role of phosphorylation in regulating organelle motility have remained unclear and controversial. A series of kinases, including PKA, PKC, and PKG, were shown to have no effect on kinesin-dependent axonal transport, whereas phosphorylation of kinesin by PKA was proposed to inhibit fast axonal transport and kinesin binding to membrane organelles. Glycogen synthase kinase 3β (GSK3β) phosphorylates KLC and kinesin-based motility is inhibited by perfusion of active GSK3β into squid axons. An inhibitory effect of CaMKII in disrupting KIF17-Mint1 association in vitro has recently been reported. However, it was not until recently that the physiological role of phosphorylation in axonal transport was addressed in vivo (Horiuchi, 2007), in which JNK-mediated phosphorylation inhibits the kinesin-1-JIP1 adaptor interaction (Toda, 2008).
This study has revealed a critical role of UNC-76 phosphorylation during axonal transport, which is likely elicited at the motor-cargo interface. Several lines of evidence support this notion. First, unc-51 genetically interacts with the kinesin-1 adaptor unc-76 in axonal transport in vivo. Second, biochemical experiments show that the association of UNC-76 and Syt-1 is mediated by UNC-51-dependent phosphorylation of UNC-76. Third, FRET analysis confirms the direct association between UNC-76 and Syt-1 in cells, which is attenuated by inhibiting UNC-51 kinase activity. Finally, the SV transport defects of unc-76 mutants are rescued in vivo by phosphomimetic UNC-76, but not by phosphodefective UNC-76 (Toda, 2008).
Based on these findings, a model is proposed in which adaptor phosphorylation is a key regulatory step that maintains motor-cargo association within axons and leads to efficient SV transport. Upon phosphorylation by UNC-51 kinase, UNC-76 displays an increased affinity to SV membrane proteins such as Syt-1. Attenuation of UNC-51 kinase activity would reduce the affinity of UNC-76 for SV membrane proteins and cause the dissociation of SV cargoes from the motor complexes. In this model, motor-cargo affinity could also be reduced by dephosphorylation of UNC-76, although such regulatory factor is yet to be identified. Attenuation of UNC-51 activity or activation of phosphatase activity could explain the mechanism of SV cargo detachment from the kinesin motors. Additional work is needed to determine how UNC-51 kinase activity is spatially and temporally controlled to regulate axonal transport. It is notable that both UNC-51 and UNC-76 are undetectable at NMJ of the wild-type third instar larvae, suggesting a spatial control of motor-cargo dissociation at the axonal termini (Toda, 2008).
The proposed mechanism can explain the transport of a subset of SVs. Only ~20% of SVs appear to colocalize with UNC-76 in wild-type segmental nerves (SGNs), suggesting that an additional motor/adaptor system, such as kinesin-3, is responsible for carrying the rest of the SVs at the larval stage. Indeed, a subpopulation of SVs successfully reaches the synapses in unc-51 mutant NMJs, although these synaptic boutons are smaller in size and fewer in number. It is also likely that UNC-51/UNC-76/kinesin-1 activity is dispensable for loading SVs onto the motor complexes at cell bodies, because SV aggregates are found within axons and distant from cell bodies in these mutants. Thus, the UNC-51/UNC-76/kinesin-1 complex seems important for maintaining motor-cargo association within axons rather than being responsible for initial cargo loading. Alternatively, maternally deposited unc-51 or Khc may contribute to partial transport of SVs into axons, and the potential role of UNC-51/UNC-76/kinesin-1 complex in initial cargo loading may be masked in the analysis of unc-51 mutants (Toda, 2008).
Although this study clearly demonstrates that phosphorylation of UNC-76 by UNC-51 kinase is critical for SV transport, the phosphomimetic UNC-76 transgene failed to rescue defective SV transport in unc-51 mutants. This implies that phosphorylation of UNC-76 is not sufficient for SV transport, and suggests that additional targets of UNC-51 phosphorylation are necessary for proper SV transport. Both KHC and KLC are phosphoproteins, and unc-51 interacts genetically with Klc. Therefore, additional transport components, including KHC and KLC, need to be tested as candidate substrates for UNC-51 kinase in order to understand the whole picture of unc-51-mediated axonal transport machinery (Toda, 2008).
It is unclear how loss of unc-51 results in aggregation of the kinesin motor complex. Biophysical studies show that cargo binding to the kinesin tail domain is required to unfold kinesin molecules and to activate their motor function on MTs, suggesting that a failure of motor-cargo assembly could cause stalling and aggregation of kinesin motors. With respect to the kinesin motor activation, a recent report has addressed a novel role for UNC-76/FEZ1 in kinesin motor unfolding and thus activation (Blasius, 2007). In this model, two scaffolding/adaptor proteins, JIP1 and UNC-76/FEZ1, induce a step-wise conformational change in kinesin-1, leading to its full activation as a motor in vitro. In good agreement with this model, loss of unc-76 results in disorganized localization of SVs and KHC in vivo. Taken together with the finding that the phosphomimetic UNC-76 is capable of rescuing axonal transport defects in unc-76 mutants, it is possible that UNC-76/FEZ1 not only serves as a motor-cargo linker, but also functions as a kinesin-1 activator in a phosphorylation-dependent manner. Thus, this phosphorylation-dependent regulation of adaptors may address an additional mechanism for controlling kinesin-1 activity that is essential for axonal transport (Toda, 2008).
Although the unc-51 and unc-76 pathways could cooperatively activate kinesin-1 activity, it is unlikely that loss of unc-51 leads to complete loss of Khc activity. In unc-51 mutants, mitochondrial transport is partially attenuated, but the majority of mitochondria are still transported, suggesting that the overall activity of KHC, a major motor for mitochondrial transport, is preserved. This view is supported by immunohistochemical evidence, which shows that a subpopulation of KHC is present in aggregates, whereas the rest of KHC was distributed throughout the axons. This suggests that a part of KHC activity may be unaffected in unc-51 mutants, and that the UNC-76-independent population of KHC is functionally active and participate in the transport of other cargoes. Although UNC-51 forms a complex with KHC via UNC-76 and KHC distribution is altered in unc-51 mutants, which strongly suggests a functional interaction between UNC-51 and KHC, it is possible that UNC-51 regulation of kinesin-1 activity is mediated through UNC-76 or an additional factor such as KLC, and the effect of UNC-51 on the KHC motor may be indirect. In this regard, it is suggestive that unc-76 and unc-51 show a clear genetic interaction, whereas Khc and unc-51 do not exhibit an apparent genetic interaction (Toda, 2008).
This model of phosphorylation-dependent regulation of motor-cargo assembly could be extended to include additional adaptors or cargo vesicles. UNC-14, a protein that interacts with UNC-51, has recently been reported to play a role in kinesin-1-dependent axonal transport in C. elegans (Sakamoto, 2005). UNC-14 might serve as an adaptor for the kinesin motor complex to regulate motor-cargo affinity in an UNC-51-dependent manner. In addition, unc-51 mutations also result in aggregation of vesicles positive for UNC-5, a Netrin/UNC-6 receptor (Ogura, 2006). Again, affinity between the UNC-5-positive vesicles and their respective motor complexes might be regulated by UNC-51-dependent phosphorylation (Toda, 2008).
Previous studies in worms and mice, as well as this study, addressed a role of unc-51 in axon formation. It remains to be studied whether this model could be extended to explain the regulation of membrane components necessary for axon formation, in which the assembly of axonal membranes with the corresponding motors may be mediated via unc-51-dependent phosphorylation (Toda, 2008).
Recent studies identified unc-51 as a homolog of atg1, which plays a role in autophagy, a catabolic cellular process responsible for bulk degradation of proteins and organelles, particularly when cells are under nutrient-deprived conditions. Disruption of the autophagy genes atg5 or atg7 in mouse brains results in neuronal cell death, which accompanies intracellular accumulation of ubiquitin-positive aggregates. Neither ubiquitin-positive aggregates nor symptoms of cellular death were observed in unc-51 mutant SGNs, suggesting that the role of unc-51/atg1 in axonal transport is distinct from its role in autophagy, which is induced under nutrient-deficient conditions. Although autophagy critically depends on intracellular vesicle transport, and UNC-51 kinase activity seems to be required for autophagy induction, a link between axonal transport and autophagy remains to be studied (Toda, 2008).
In conclusion, this study identifies a novel regulatory step for axonal transport that depends on the UNC-51 kinase-mediated phosphorylation of a kinesin adaptor. Further studies on the regulation of unc-51 activity will provide a better understanding of axonal transport, as well as dynamic neuronal control of synaptic development and plasticity (Toda, 2008).
Efficient synaptic transmission requires the apposition of neurotransmitter release sites opposite clusters of postsynaptic neurotransmitter receptors. Transmitter is released at active zones, which are composed of a large complex of proteins necessary for synaptic development and function. Many active zone proteins have been identified, but little is known of the mechanisms that ensure that each active zone receives the proper complement of proteins. This study used a genetic analysis in Drosophila to demonstrate that the serine threonine kinase Unc-51 acts in the presynaptic motoneuron to regulate the localization of the active zone protein Bruchpilot opposite to glutamate receptors at each synapse. In the absence of Unc-51, many glutamate receptor clusters are unapposed to Bruchpilot, and ultrastructural analysis demonstrates that fewer active zones contain dense body T-bars. In addition to the presence of these aberrant synapses, there is also a decrease in the density of all synapses. This decrease in synaptic density and abnormal active zone composition is associated with impaired evoked transmitter release. Mechanistically, Unc-51 inhibits the activity of the MAP kinase ERK to promote synaptic development. In the unc-51 mutant, increased ERK activity leads to the decrease in synaptic density and the absence of Bruchpilot from many synapses. Hence, activated ERK negatively regulates synapse formation, resulting in either the absence of active zones or the formation of active zones without their proper complement of proteins. The Unc-51-dependent inhibition of ERK activity provides a potential mechanism for synapse-specific control of active zone protein composition and release probability (Wairkar, 2009).
A large-scale anatomical screen was performed to identify mutants where not every glutamate receptor cluster is apposed to Bruchpilot. Mutants with a global decrease in Brp or DGluRIII across the NMJ were put aside, and instead focus was placed on mutants in which Brp was absent from a subset of synapses. Such mutants were identified by the presence of glutamate receptor clusters unapposed to Bruchpilot puncta. In this screen, mutants were identified in unc-51 (Wairkar, 2009).
In the unc-51 mutant many DGluRIII clusters are unappposed to Brp. Such misapposition could reflect either DGluRIII clusters unapposed to active zones, or receptor clusters apposed to abnormal active zones that do not contain Brp. The ideal experiment to distinguish between these possibilities would be to stain for other presynaptic active zone proteins. Unfortunately the only other such protein that can be visualized in Drosophila is the calcium channel Cacophony, and since its localization depends on Brp this experiment is not be informative. Nonetheless, two results strongly suggest that a subset of glutamate receptors is apposed to abnormal active zones. First, the decreased density of DGluRIII clusters observed via confocal microscopy approximates the decrease in active zone density observed via electron microscopy. If many DGluRIII clusters were unapposed to active zones, then a more dramatic decrease in active zone density would be expected. Second, ultrastructural analysis demonstrates a decrease in the proportion of active zones containing T-bars. Brp is not necessary for the formation of active zones, but is required for the localization of T-bars to active zones. If the absence of Brp were due to the absence of the entire active zone, then each active zone would contain Brp and a normal ratio of T-bars/active zones would be predicted. Instead, the decrease in T-bars/active zone is consistent with the presence of active zones missing Brp and, hence, lacking T-bars. Therefore, it is concluded that Unc-51 is required for the high fidelity of active zone assembly, ensuring that Brp is present at every active zone (Wairkar, 2009).
In addition to the presence of abnormal synapses in the unc-51 mutant, there is also a decrease in the number and density of synapses. It is speculated that the decrease in synaptic density and the presence of abnormal synapses may be related phenotypes that differ in severity. In this view, Unc-51 promotes synapse formation. In its absence, active zone assembly would be less efficient, resulting in either the formation of abnormal active zones missing crucial proteins such as Brp, or in more severe cases leading to complete failure of active zone assembly and, hence, the absence of a synapse. The complete suppression of both the synaptic density and apposition phenotypes by mutation of the downstream target ERK is consistent with these phenotypes sharing an underlying mechanism. As expected, this defect in the number and proper assembly of synapses leads to a dramatic decrease in synaptic efficacy (Wairkar, 2009).
In addition to these synaptic defects, the unc-51 mutant also has a smaller NMJ and accumulations of synaptic material in the axons, suggesting defects in axonal transport. One mechanism that could link a small NMJ with defective transport is synaptic retraction, in which entire presynaptic boutons or branches retract leaving a footprint of postsynaptic proteins. However, no such footprints were observed in the unc-51 mutant, so this is not the cause of the small NMJ. Synaptic growth requires the retrograde transport of a BMP signal to the nucleus, however this study no change in the levels of phosphorylated MAD in motoneuron nuclei, suggesting that this is not a likely cause of the growth defect. Finally, in worms and mice Unc-51 is required for axon outgrowth, which may be somewhat analogous to defects in NMJ growth in Drosophila. However, to form an NMJ the axon must navigate out of the ventral nerve cord and cross a wide expanse of muscle before reaching its target and forming a junction. Since no defects were observed in the pattern of neuromuscular innervation, it is unlikely that a generic defect in axon outgrowth is responsible for the small NMJs. The apparent axonal transport defect is consistent with findings from mammals suggesting a function for Unc-51 in regulating axon transport. The role of Unc-51 for transport was not investigated, but note that it was possible to genetically separate the axonal transport and synapse development phenotypes, so the transport phenotypes may not be primary cause of the synaptic defects (Wairkar, 2009).
These data support the model that Unc-51 inhibits ERK activation to promote proper active zone development. In the unc-51 mutant a modest increase was observed in the levels of activated ERK, demonstrating that Unc-51 is a negative regulator of ERK activation in vivo. This increased ERK activity is responsible for the defects in active zone formation. Double mutants between unc-51 and the ERK hypomorph rl1 completely suppress the synapse density and apposition phenotypes of the unc-51 mutant, and restore synaptic strength to wild type levels. Hence, ERK is required for the synaptic phenotypes observed in the unc-51 mutant. The axonal transport defects were not suppressed in the double mutant, so Unc-51 must act through other pathways as well. In mammalian cells Unc-51 can downregulate ERK by inhibiting the binding of a scaffolding protein to the FGF receptor. To date, no receptor tyrosine kinase has been identified that regulates active zone formation in Drosophila. Future studies to characterize the mechanism by which Unc-51 inhibits ERK in Drosophila motoneurons may provide clues towards identification of such a pathway. In addition, it is unclear how ERK regulates active zone formation. A previous study demonstrated that phospho-ERK localizes to the active zone, which would suggest a direct mechanism. Unfortunately, these localization findings could not be replicated. The same study demonstrated that the transgenic expression of a constitutively active ras or a gain-of-function ERK allele both lead to an increase in the number of synaptic boutons, which is not consistent with the current finding of a smaller NMJ. Active zone structure and number were not assessed. It is speculated that the global activation of ERK may result in different phenotypes than relief of Unc-51 inhibition of ERK, which could show temporal and spatial specificity (Wairkar, 2009).
In mammalian and Drosophila neurons, release probability varies across release sites formed by a single neuron. One potential mechanism would be the differential localization or activity of core active zone proteins. In Drosophila, Bruchpilot is an excellent candidate for such a protein. It is required for the localization of calcium channels to the active zone, so changes in its localization or function would impact calcium influx and, hence, release probability at an active zone. The unc-51 mutant demonstrates that signaling pathways can differentially regulate the localization of Brp to individual release sites within a single neuron. As such, the Unc-51/Erk signaling pathway is a candidate mechanism to regulate active zone protein composition and release probability in a synapse-specific manner (Wairkar, 2009).
Autophagy is a membrane-mediated degradation process of macromolecule recycling. Although the formation of double-membrane degradation vesicles (autophagosomes) is known to have a central role in autophagy, the mechanism underlying this process remains elusive. The serine/threonine kinase Atg1 has a key role in the induction of autophagy. This study shows that overexpression of Drosophila Atg1 promotes the phosphorylation-dependent activation of the actin-associated motor protein myosin II. A novel myosin light chain kinase (MLCK)-like protein, Spaghetti-squash activator (Sqa), was identified as a link between Atg1 and actomyosin activation. Sqa interacts with Atg1 through its kinase domain and is a substrate of Atg1. Significantly, myosin II inhibition or depletion of Sqa compromised the formation of autophagosomes under starvation conditions. In mammalian cells, it was found that the Sqa mammalian homologue zipper-interacting protein kinase (ZIPK) and myosin II had a critical role in the regulation of starvation-induced autophagy and mammalian Atg9 (mAtg9; see Drosophila Atg9) trafficking when cells were deprived of nutrients. These findings provide evidence of a link between Atg1 and the control of Atg9-mediated autophagosome formation through the myosin II motor protein (Tang, 2011).
Myosin II is a conventional two-headed myosin composed of two heavy chains, two essential light chains, and two regulatory light chains. Myosin II activation is regulated by the phosphorylation of its regulatory light chain via MLCKs. Rho GTPase and Rho kinase have been implicated in the regulation of myosin activation. However, this study found that neither RNA-mediated knockdown of dRok nor mutations in Rho1 or dRhoGEF2 could suppress the Atg1-induced wing defects. Instead, it was found that depletion of Sqa rescued Atg1-induced wing defects. This epistasis analysis showed that Sqa functioned downstream of Atg1. Moreover, it was found that Sqa but not Atg1 could directly phosphorylate Spaghetti squash (Sqh) in the in vitro kinase assay, suggesting that Atg1 stimulates myosin activity via Sqa. Importantly, Atg1 phosphorylates and interacts with Sqa, indicating that Atg1-Sqa functions in a kinase cascade to regulate myosin II activation. Moreover, Atg1 has been found to have a critical role in the regulation of autophagy induction under stress conditions in yeast, Drosophila, and mammalian cells. These results provide the first evidence that nutrient starvation stimulates myosin II activation in an Atg1-Sqa-dependent manner. Most significantly, a dramatic decrease was found in the size and number of autophagosomes in cells expressing Sqa-T279A, Sqa-RNAi, and SqhA20A21 on nutrient deprivation, indicating that Atg1-Sqa-mediated actomyosin activation has a critical role in autophagy (Tang, 2011).
The kinase domain of Sqa is also highly homologous to that of the mammalian DAPK family proteins. Recent studies have indicated that DAPK1 regulates autophagy through its association with MAP1B and Beclin1, or by modulating the Tor signalling pathway. As DAPK family proteins also regulate myosin II phosphorylation, one might speculate that Sqa may be the Drosophila counterpart of DAPK protein. Indeed, although overexpression of Sqa does not induce cell death, Sqa shares several characteristics with DAPK3/ZIPK. First, unlike MLCK family proteins, both Sqa and ZIPK contain an amino-terminal kinase domain that has 42% sequence identity and 61% similarity. Moreover, like ZIPK, recent sequence analysis from FlyBase identified a Sqa isoform that also contains a leucine-zipper domain. Second, as phosphorylation of Thr-265 in ZIPK is essential for its kinase activity, this study found that Atg1 phosphorylates Sqa at the corresponding Thr-279, and is critical for Sqa activity. Third, just as Sqa specifically associates with kinase-inactive Atg1, the results indicate a similar interaction between ZIPK and Ulk1. Importantly, depletion of Sqa and ZIPK resulted in autophagic defects in response to nutrient deprivation. These findings together suggest that ZIPK may act as a mammalian homolog of Sqa during starvation-induced autophagy. Further investigation is needed to determine whether the mammalian Atg1 (Ulk1) directly phosphorylates ZIPK at Thr-265, and the role of this regulation in autophagy (Tang, 2011).
In autophagy, the source of the autophagosomal membrane and dynamics of autophagosome formation are fundamental questions. Studies in yeast and mammalian cells have identified several intracellular compartments as potential sources for the PAS (also termed isolation membrane/phagophore). Formation of PI(3)P-enriched ER subdomains (omegasomes) has been reported during nutrient starvation and autophagy induction, and a direct connection has been observed between ER and the phagophore using the 3D electron tomography. In addition, recent studies in yeast cells have suggested Atg9 and the Golgi complex have a role in the formation of autophagosomes. It has been proposed that the integral membrane protein Atg9 may respond to the induction signal in promoting lipid transport to the forming autophagosomes. The mAtg9 has been found to localize on the TGN and the endosomes in nutrient-rich conditions and translocate to LC3-positive autophagosomes on nutrient deprivation. Although several proteins, including Ulk1, mAtg13, and p38IP, have been found to regulate starvation-induced mAtg9 trafficking, the molecular motor that controls the movement of mAtg9 between different subcellular compartments remains unknown (Tang, 2011).
The finding that myosin II redistributes from peripheral to the perinuclear region of cells on starvation suggests that myosin II has a role in membrane trafficking. In fact, it has been reported that myosin II is required for the trafficking of major histocompatibility complex (MHC) class II molecules and antigen presentation in B lymphocytes. Myosin II has also been found to be involved in the protein transport between ER and Golgi. This study has shown that there here is a molecule link between mAtg9 and the actomyosin network, indicating that myosin II may function as a motor protein for mAtg9 trafficking during early autophagosome formation. In conclusion, this work has unravelled a regulatory mechanism between Atg1 activity and the Atg9-mediated formation of autophagosomes. Further studies are needed to determine the involvement of this signalling process in other stress-induced or developmentally regulated autophagy (Tang, 2011).
Members of the evolutionary conserved Ser/Thr kinase Unc-51 family are key regulatory proteins that control neural development in both vertebrates and invertebrates. Previous studies have suggested diverse functions for the Unc-51 protein, including axonal elongation, growth cone guidance, and synaptic vesicle transport. This work investigated the functional significance of Unc-51-mediated vesicle transport in the development of complex brain structures in Drosophila. It is shown that Unc-51 preferentially accumulates in newly elongating axons of the mushroom body, a center of olfactory learning in flies. Mutations in unc-51 cause disintegration of the core of the developing mushroom body, with mislocalization of Fasciclin II (Fas II), an IgG-family cell adhesion molecule important for axonal guidance and fasciculation. In unc-51 mutants, Fas II accumulates in the cell bodies, calyx, and the proximal peduncle. Furthermore, it was shown that mutations in unc-51 cause aberrant overshooting of dendrites in the mushroom body and the antennal lobe. Loss of unc-51 function leads to marked accumulation of Rab5 and Golgi components, whereas the localization of dendrite-specific proteins, such as Down syndrome cell adhesion molecule (DSCAM) and No distributive disjunction (Nod), remains unaltered. Genetic analyses of kinesin light chain (Klc) and unc-51 double heterozygotes suggest the importance of kinesin-mediated membrane transport for axonal and dendritic development. Moreover, these data demonstrate that loss of Klc activity causes similar axonal and dendritic defects in mushroom body neurons, recapitulating the salient feature of the developmental abnormalities caused by unc-51 mutations. It is concluded Unc-51 plays pivotal roles in the axonal and dendritic development of the Drosophila brain. Unc-51-mediated membrane vesicle transport is important in targeted localization of guidance molecules and organelles that regulate elongation and compartmentalization of developing neurons (Mochizuki, 2011).
Active transport and delivery of molecular and cellular components from the soma to the distinct cytoplasmic compartments is critical not only for synaptic function in mature neurons but also for axonal elongation and guidance in developing neurons. The kinesin-mediated anterograde transport plays a major role in the active traffic in the developing neurons, delivering a wide range of cargos along the axon, including synaptic vesicles, mitochondria, cytoskeletal elements, and mRNAs (Mochizuki, 2011).
This work has demonstrated preferential expression of Unc-51 and kinesin motor proteins in larval MBs, and has shown that loss of unc-51 activity causes severe defects in kinesin-mediated transport in developing MB neurons, while dynein/dynactin-mediated retrograde transport is unaffected in unc-51 mutant MBs. In addition, loss of unc-51 activity results in disintegration of axonal bundles and aberrant extensions of dendrites in both MB and AL neurons. These results suggest that unc-51 is essential for the development of brain neurocircuitries, participating in molecular and/or cellular mechanisms that regulate the formation of the complex structures such as MBs. Indeed, the results demonstrate that unc-51 is essential for the specific intracellular localization of axonal fasciculation and guidance molecules such as Fas II (Mochizuki, 2011).
Although previous studies have demonstrated that unc-51 has diverse functions in developing neurons, this analyses of the Klc+/- unc-51+/- double heterozygotes clearly demonstrate that suppression of kinesin-mediated transport results in dendritic overextensions. Concomitant suppression of unc-51 and Klc also causes axonal transport defects that are reminiscent of unc-51 mutants. Furthermore, the double heterozygotes exhibit mislocalization of Fas II in both the calyx and the proximal peduncle. This recapitulates the salient phenotype of unc-51-/- mutants, and argues that defective kinesin-mediated transport is the major molecular process that underlies the developmental defects in the unc-51 mutants. In contrast, Klc+/- unc-51+/- double heterozygotes failed to exhibit a full range of the unc-51 mutant phenotypes. Although the possibility cannot be excluded that other molecular processes are involved in the unc-51 mutant phenotypes, the weaker phenotypes of the double heterozygotes might be accounted by partial suppression of the gene activity given that wild-type alleles are retained at a half dosage for both genes. Furthermore, the result that Klc null mutant clones exhibited dendritic and axonal defects that were reminiscent of the unc-51 mutant clones clearly confirms the importance of the kinesin-mediated transport in brain development. It is also noteworthy that, as with Klc mutant clones, Khc null MB clones exhibit similar yet more pronounced defects, although the critical requirement of the Khc activity for neuroblast division hampers a precise assessment of Khc function in axodendritic development. These results as a whole highlight the importance of kinesin-mediated vesicle transport in the development and wiring of complex networks in the brain (Mochizuki, 2011).
Previous studies have demonstrated that unc-51 plays an essential role in axonal transport by mediating the assembly of the cargos and the kinesin motor proteins. In unc-51 mutants, membrane vesicle transport is severely affected, resulting in accumulation of synaptic vesicles in the larval segmental nerves. Loss of unc-51 activity also causes aggregation of Rab5-positive membranes in the segmental nerves. Notably, as in the mutant MBs, kinesin motors are accumulated in the mutant segmental nerves while overall mitochondrial localization was unaffected. Genetic studies have shown that the wild-type but not a kinase-deficient form of the unc-51 transgene rescues the transport defect in synaptic vesicles in the mutant segmental nerves. Moreover, the kinase activity of Unc-51 is critical for phosphorylation of an adaptor molecule, Unc-76/FEZ, which mediates the assembly between synaptic vesicle cargos and the kinesin motor complex. The finding that the dendritic and axonal defects in MB neurons are rescued by the wild-type but not by the kinase-deficient unc-51 transgene suggests that a similar phosphorylation-dependent regulatory mechanism is involved in the intracellular transport in developing MB neurons. In line with this, it is noteworthy that Unc-76 is preferentially expressed in developing MB neurons, in which it colocalizes with Unc-51 and kinesin motor proteins in the core fibers. Intriguingly, both Unc-51 and Unc-76 are downregulated in the mature neurons that surround the core fibers as they mature and shift to the peripheral layers. Concomitantly, both Khc and Klc are downregulated in the mature MB neurons, suggesting dynamic control of the expression of the molecular components that mediate active vesicle transport in developing MB neurons (Mochizuki, 2011).
Recent genetic studies on the dendritic development of Drosophila larval sensory neurons showed that the microtubule motor protein dynein controls dendritic branching by directing polarized intracellular vesicle trafficking. Dynein is also necessary for the dendrite-specific localization of Golgi outposts, which secretory pathway plays critical roles in dendritic growth and branching. These studies also showed that Rab5 was essential to normal dendritic branching, via its role in controlling endosomal trafficking. The current results show that mutations of unc-51 leads to aberrant accumulations of Rab5-containing endosomes and Golgi components in developing larval MBs. In contrast, polarized dendritic transport of Dscam is not altered in the unc-51 mutant MBs, implying that retrograde dynein/dynactin-mediated transport remains intact in the mutant MB neurons. Moreover, another dendritic molecule, Nod, is correctly targeted to the calyx, clearly indicating that unc-51 is not required for polarized retrograde transport mediated by dynein/dynactin in developing MB neurons. These results are consistent with previous observations that unc-51 fails to interact with retrograde motor genes such as dynein heavy chain and Lissencephaly-1 in segmental nerves (Mochizuki, 2011).
In contrast, previous studies found that khc mutants showed dendritic branch abnormalities that were almost identical to those of dynein light intermediate chain (Dlic) mutants, with reduced arbors and a marked shift in branching activity in the proximal area within the arbors. In the khc mutants, the dynein complexes become aggregated distally, suggesting that kinesin plays a role in recycling dynein proximally after it has carried its organelle cargo distally. These phenotypes seem to contrast with those observed in unc-51 mutant MB neurons, which show dendritic overextensions with normal dynein/dynactin-mediated transport. The exact mechanisms underlying these discrepancies are unknown, but this might suggest different regulatory processes for kinesin-mediated transport that operate in the distinct cellular contexts of the peripheral sensory neurons and MB neurons. It was showm that unc-51 mutation resulted in varying degrees of axonal membrane defects that were dependent on the cargo. It is possible that Unc-51 differentially regulates the transport of specific cargo subsets by phosphorylation of distinct groups of adaptor proteins in different cell types (Mochizuki, 2011).
Studies in Drosophila identified unc-51 as a homolog of the yeast atg1, and suggested that Unc-51 kinase activity is required for the induction of autophagy. It has been shown that autophagy positively regulates synapse development at the Drosophila neuromuscular junction. Mutations in autophagy genes including atg1/unc-51, caused significant reduction in terminal branching of the segmental motoneurons, with reduced numbers of boutons. In contrast, this study has shown that single-cell analysis of AL-PNs shows that loss of unc-51 activity results in an increase in the number of the calyx branches. The results also demonstrate that the distribution of an autophagy marker is not altered between the wild-type and the unc-51 mutant MBs. These results argue against autophagy as an underlying mechanism of the axodendritic abnormalities in the unc-51 mutant larval brain, and are consistent with a previous report that autophagy is not involved in Unc-51-mediated regulation of axonal transport (Mochizuki, 2011).
In C. elegans, mutations in unc-51 cause diverse axonal defects, including premature termination, abnormal trajectories, and extra axon branches. Developing neurites accumulate abnormal vesicles and cisternae, suggesting underlying defects in membrane vesicle trafficking. Intriguingly, Unc-51 directly interacts with Unc-14, a RUN domain protein, to regulate axonal elongation and guidance, and mutations in unc-14 cause neurite growth and guidance defects that are very similar to those of unc-51. Unc-14 regulates vesicle transport and localization by binding to Unc-16/JIP3/JSAP1, which is a cargo adaptor for the kinesin motor proteins. Recent studies have shown that RUN domain proteins function as effectors of Rap and Rab GTPases in the control of membrane trafficking, highlighting the importance of vesicle trafficking in the regulation of axonal growth and guidance. Several studies have suggested that Unc-51 plays an essential role in the delivery of specific receptors for axonal guidance molecules. Together with Unc-14, Unc-51 regulates the subcellular localization of the Netrin receptor/Unc-5 in C. elegans. Thus, in unc-14 and unc-51 mutants, the Netrin receptor accumulates in neural cell bodies rather than at axonal termini, causing severe guidance defects in the DD/DV neuron. Unc-51 also interacts with the kinesin-related Vab-8 protein, which regulates anterior-posterior migration of C. elegans mechanosensory neurons through the regulation of another Netrin receptor Unc-40/Dcc and the Slit receptor Sax-3/Robo. Vab-8 controls cell-surface expression of Sax-3/Robo in the growth cones of touch neurons through interaction with Unc-73/Trio, a guanine nucleotide exchange factor for Rac. Consequently, peptide-mediated interference with the Vab-8 and Unc-51 interaction in worm neurons blocked axonal outgrowth and posteriorly directed guidance (Mochizuki, 2011).
In mouse, Unc51.1/Ulk1 is expressed in granule cells in the cerebellar cortex, and retroviral infection of immature granule cells with a dominant-negative Unc51.1 caused inhibition of neurite outgrowth both in vitro and in vivo. Subsequent molecular studies showed that Unc51.1 binds to SynGAP and Syntenin, the latter of which, in turn, binds Rab5 GTPase to tether the Unc51.1/SynGAP/Rab5 complex to the vesicular membrane. Immunoelectron microscopy of granule cells provided evidence that Unc51.1 indeed associates with membrane vesicles. Moreover, SynGAP stimulates the GTPase activity of Rab5, and overexpression of SynGAP in cultured cerebellar granule neurons leads to truncated neurites and disorganized vesicular compartments. This suggests that the Unc51.1-containing protein complex governs axonal elongation and pathfinding by modulating the Ras-like GTPase signaling pathway and the Rab5-mediated endocytic pathway in developing neurons (Mochizuki, 2011).
The importance of Unc-51 in the regulation of vesicle trafficking is further supported by the finding that suppression of Unc-51 activity leads to increased neurite branch formation and shortened axons in cultured mouse dorsal root ganglia neurons. Both Unc51.1 and Unc51.2 are localized to vesicular structures in growth cones in sensory axons, in which Unc51.1 promotes endocytosis of the neurotrophic tyrosine kinase receptor Ntrk1/TrkA through a non-clathrin mediated pathway, presumably through the interaction of Unc51.1 with SynGAP and Rab5. Moreover, Unc51.1 also interacts with the Golgi-associated ATPase enhancer of 16 kD (Gabarapl2/Gate-16), which is an essential factor for intra-Golgi transport\. Unc51.1 also interacts with the gamma-2 subunit of the GABA-A receptor associated protein (GABARAP), which is again involved in the regulation of receptor trafficking. Together with the current findings in the Drosophila brain, these studies highlight the functional importance of the Unc-51 family proteins in the endocytic processes that regulate diverse signaling events during axonal elongation, fasciculation, and guidance. Loss of the Unc-51 activity is likely to perturb the trafficking of multiple types of post-Golgi vesicles and lead to severe disruption of the controlled delivery of essential axonal growth/guidance receptors to the different compartments of growing neurons. Elucidation of the exact molecular components that are involved in Unc-51-mediated regulation of vesicle transport is an important subject for future studies. It is envisaged that studies in Drosophila will continue to provide critical insights into the conserved molecular mechanisms of coordinate regulation of membrane vesicle trafficking and axon growth/guidance in developing neurons (Mochizuki, 2011).
UNC-51 is a serine/threonine protein kinase conserved from yeast to humans. The yeast homolog Atg1 regulates autophagy (catabolic membrane trafficking) required for surviving starvation. In C. elegans, UNC-51 regulates the axon guidance of many neurons by a different mechanism than it and its homologs use for autophagy. UNC-51 regulates the subcellular localization (trafficking) of UNC-5, a receptor for the axon guidance molecule UNC-6/Netrin; however, the molecular details of the role for UNC-51 are largely unknown. This study reports that UNC-51 physically interacts with LET-92, the catalytic subunit of serine/threonine protein phosphatase 2A (PP2A-C), which plays important roles in many cellular functions. A low allelic dose of LET-92 partially suppresses axon guidance defects of weak, but not severe, unc-51 mutants, and a low allelic dose of PP2A regulatory subunits A (PAA-1/PP2A-A) and B (SUR-6/PP2A-B) partially enhance the weak unc-51 mutants. It was also found that LET-92 can work cell-non-autonomously on axon guidance in neurons, and that LET-92 colocalizes with UNC-51 in neurons. In addition, PP2A dephosphorylates phosphoproteins that have been phosphorylated by UNC-51. These results suggest that, by forming a complex, PP2A cooperates with UNC-51 to regulate axon guidance by regulating phosphorylation. This is the first report of a serine/threonine protein phosphatase functioning in axon guidance in vivo (Ogura, 2010).
Autophagy, a lysosome-dependent degradation mechanism, mediates many biological processes, including cellular stress responses and neuroprotection. This study demonstrates that autophagy positively regulates development of the Drosophila larval neuromuscular junction (NMJ). Autophagy induces an NMJ overgrowth phenotype closely resembling that of highwire (hiw), an E3 ubiquitin ligase mutant. Moreover, like hiw, autophagy-induced NMJ overgrowth is suppressed by wallenda (wnd) and by a dominant-negative c-Jun NH2-terminal kinase (bskDN). Autophagy promotes NMJ growth by reducing Hiw levels. Thus, autophagy and the ubiquitin-proteasome system converge in regulating synaptic development. Because autophagy is triggered in response to many environmental cues, these findings suggest that it is perfectly positioned to link environmental conditions with synaptic growth and plasticity (Shen, 2009).
Autophagy involves multiple steps, including induction, autophagosome formation, fusion of autophagosomes with lysosomes, and recycling of autophagy components. Disrupting any of these steps impairs autophagy. Several highly conserved ATG genes encoding core components of the autophagy machinery have been identified in yeast. Mutations in genes, including atg1, -2, -6, and -18, have also been isolated and characterized in Drosophila. To assess the role of autophagy in NMJ development, the effects were examined of mutations in atg genes, whose normal functions span the entire process: atg1 is defective in autophagy induction, atg6 is defective in autophagosome formation, and atg2 and -18 are defective in retrieval of other ATG proteins from autophagosomes. Regardless of the step impaired, all of these atg mutants exhibited significant reduction in NMJ size. These results demonstrate that a basal level of autophagy is required to promote NMJ development (Shen, 2009).
Overexpression of atg1+ is sufficient to induce high levels of autophagy in larval fat bodies and salivary glands. If autophagy is a positive regulator of NMJ development, an increase in autophagy might enhance synaptic growth. Consistent with previous studies, panneuronal overexpression of UAS-atg1+ under the control of C155-Gal4 or elav-Gal4 drivers induced high levels of autophagy in the nervous system, as indicated by increased staining with LysoTracker, an acidophilic dye which has been used to assess autophagy by labeling acidic structures, including lysosomes. Under these conditions, bouton number increased more than twofold. To further verify that this NMJ overgrowth was caused by elevated autophagy rather than to some other effect of atg1+ overexpression, whether mutations in other atg genes suppress this phenotype was examined. For this purpose, a null allele of atg18 (atg18δ) was generated. Removal of one copy of atg18+ had no affect on NMJ growth in an otherwise wild-type background but significantly suppressed NMJ overgrowth caused by atg1+ overexpression. Removal of both copies of atg18+ conferred almost complete suppression. Therefore, NMJ overgrowth caused by atg1+ overexpression is primarily caused by elevated levels of autophagy (Shen, 2009).
As a further test, NMJ morphology was examined after feeding larvae with rapamycin, which induces autophagy by inhibiting TOR (target of rapamycin), the key negative regulator of autophagy. Wild-type larvae fed rapamycin exhibited striking NMJ overgrowth similar to that caused by overexpressing atg1+. Rapamycin-induced NMJ overgrowth was completely suppressed by mutations in atg18. Collectively, these results demonstrate that autophagy is a key positive regulator of NMJ growth (Shen, 2009).
Wairkar (2009) observed NMJ undergrowth in atg1 mutants but did not see overgrowth with atg1+ overexpression. This discrepancy likely results from the use of different UAS-atg1+ transgenes. For example, Wairkar was able to obtain only partial (~50%) rescue of NMJ undergrowth in atg1 mutants by overexpression of their UAS-atg1rescue construct, whereas this study obtained complete rescue of this phenotype (Shen, 2009).
Atg1 has several functions unrelated to autophagy. It was found that axonal transport is disrupted in atg1-null mutants, which is a result also recently reported by Toda (2008) and Wairkar (2009). In addition, Atg1 suppresses translation by inhibiting the S6K kinase (Lee, 2007; Scott, 2007) and controls active zone density by inhibiting extracellular signal-regulated kinase (ERK) signaling (Wairkar, 2009). However, several lines of evidence indicate that these functions of Atg1 are not responsible for the NMJ phenotypes observed when Atg1 activity was altered. (1) atg2 or -18 mutants exhibited similar NMJ undergrowth but did not have defects in axonal transport. Thus, in agreement with Toda (2008), it is concluded that Atg1's role in axonal transport is distinct from its function in autophagy and NMJ growth. (2) Blocking or activating translation by overexpressing a dominant-negative S6K transgene or constitutively activated S6K transgenes by elav-Gal4 driver had little affect on NMJ growth. Moreover, coexpression of any of the three constitutively activated S6K transgenes failed to suppress NMJ overgrowth caused by atg1+ overexpression. Thus, the role of Atg1 in S6K-dependent translation does not contribute to the NMJ phenotypes associated with manipulations of Atg1. (3) An ERK mutation does not affect NMJ growth. Although this ERK mutation suppresses the deficit in active zone density in atg1 mutants, it does not suppress atg1's NMJ undergrowth phenotype (Wairkar, 2009), indicating that it is not mediated by the ERK pathway. Collectively, these results demonstrate that altered levels of autophagy are primarily responsible for the effects of Atg1 on NMJ development (Shen, 2009).
NMJ overgrowth induced by autophagy is distinctive and offers potential clues about pathways that may be involved. Formation of multiple long synaptic branches containing many small diameter boutons without any hyperbudding or satellite boutons most closely resembles the hiw phenotype, suggesting that autophagy and Hiw may function through the same pathway. Recent evidence indicates that Hiw inhibits NMJ growth by down-regulating Wnd, which in turn activates a Jun kinase encoded by bsk (basket). NMJ overgrowth in hiw is suppressed by mutations of wnd and by a dominant-negative mutation of bsk (bskDN; Collins, 2006). If the phenotypic similarity between hiw and increased autophagy reflects convergence on a common pathway, autophagy-induced NMJ overgrowth should also be suppressed by wnd and bskDN. Indeed, this is what was observed in this study. These results strongly support the idea that autophagy and Hiw converge on a Wnd-dependent MAPK signaling pathway to regulate NMJ development (Shen, 2009).
If autophagy and Hiw act via a common pathway, where do they converge? As a positive regulator of NMJ growth, autophagy could promote degradation of a negative regulator. An intriguing possibility is that Hiw is the negative regulator affected by autophagy. If a decrease in Hiw levels is responsible for NMJ overgrowth when autophagy is elevated, restoration of Hiw should suppress overgrowth. This possibility was tested by coexpressing wild-type Hiw with Atg1; Atg1-mediated NMJ overgrowth was found to be significantly suppressed. This suppression is not simply an indirect consequence of the dilution of GAL4 caused by addition of a second UAS element because coexpression of UAS-nwk+ did not suppress such NMJ overgrowth. This result also shows that Nwk (Nervous wreck), another negative regulator of NMJ growth, is not an apparent target of autophagy, as predicted by differences in phenotypes. Thus, autophagy appears to regulate NMJ growth through its effects on particular presynaptic proteins, and Hiw represents a key downstream effector (Shen, 2009).
To further test whether autophagy promotes NMJ growth by limiting Hiw, one copy of hiw+ was eliminated to determine whether this further decrease in Hiw levels enhanced the effects of atg1+ overexpression. In an otherwise wild-type background, loss of one copy of hiw+ had no affect, but it significantly enhanced atg1+-induced NMJ overgrowth. The phenotype of hiw homozygotes overexpressing atg1+ was no more extreme than hiw homozygote alone. The absence of any additive or synergistic effects further supports the hypothesis that autophagy promotes NMJ development by down-regulating Hiw (Shen, 2009).
Because Hiw antibodies do not work for immunohistochemistry, Hiw was visualized using a fully functional GFP-tagged construct to test directly whether abundance of Hiw is affected by autophagy. In an otherwise wild-type background, Hiw-GFP was strongly expressed in neurons throughout the ventral ganglion and brain lobes driven by C155-Gal4, as detected by anti-GFP staining. However, in larvae co-overexpressing atg1+, the GFP signal was reduced by ~60% relative to anti-HRP staining. This result was confirmed by Western blot analysis. Reduction of Hiw-GFP is not caused by the dilution of GAL4 by the presence of a second UAS element because coexpression of UAS-myr-RFP did not affect abundance of Hiw-GFP. These results further indicate that autophagy promotes NMJ growth by down-regulating Hiw (Shen, 2009).
These results indicate that NMJ overgrowth caused by elevated autophagy is primarily caused by reduction in Hiw. Is the converse also true? Is NMJ undergrowth in atg mutants caused by elevated levels of Hiw? To address these questions, Hiw-GFP was expressed in neurons using C155-Gal4 in various backgrounds. Hiw-GFP levels were significantly elevated in atg1 and -6 mutants compared with the controls, consistent with the idea that Hiw is down-regulated by autophagy. If this increase in Hiw is a primary cause of NMJ undergrowth in atg loss-of-function mutants, eliminating Hiw should prevent this undergrowth; i.e., mutations in hiw should be epistatic to atg mutations. Thus, NMJ morphology was examined in hiw; atg2 and hiw; atg18 double mutants, and it was found that hiw was completely epistatic, demonstrating the role of elevated levels of Hiw in NMJ undergrowth of atg mutants (Shen, 2009).
A more direct test is to determine whether overexpression of Hiw can reduce NMJ size. However, this experiment is complicated because overexpression of Hiw by a relatively weak neuronal driver (elav-Gal4) does not affect NMJ size, whereas overexpression of Hiw by a strong neuronal driver (Elav-GeneSwitch) has a modest dominant-negative effect. To determine whether increased levels of Hiw can limit NMJ growth, it appears necessary to overexpress Hiw at an intermediate level. Therefore, NMJs were examined in larvae overexpressing UAS-hiw+ via C155-Gal4. C155-Gal4/+; UAS-hiw+/+ female larvae exhibited very mild NMJ undergrowth. Stronger undergrowth was observed in C155-Gal4/Y; UAS-hiw+/+ male larvae. This difference is consistent with higher levels of C155-Gal4 expression in males than in females, owing to dosage compensation. No differences were observed in NMJ growth between C155-Gal4/+ female and C155-Gal4/Y male larvae, indicating that the undergrowth phenotypes are dependent on the levels of Hiw overexpression and not on differences in gender or expression of GAL4 alone. Thus, moderate increases in Hiw levels result in NMJ undergrowth. Furthermore, the modest NMJ undergrowth in C155-Gal4/+; UAS-hiw+/+ larvae was enhanced when one copy of atg1+, -2+, or -6+ was removed. Together, these results indicate that elevated levels of Hiw account for most of the NMJ undergrowth in atg mutants. However, excess Hiw cannot fully explain NMJ undergrowth in atg mutants because NMJ undergrowth caused by Hiw overexpression is less severe than that of atg1 and -18 mutants. Thus, when autophagy is impaired, additional negative regulators may accumulate to depress NMJ growth. It is also likely that elevated levels of Hiw target proteins other than Wnd to limit synaptic growth because loss-of-function mutations of wnd do not affect NMJ development (Shen, 2009).
Because autophagy is generally thought of as a nonselective bulk degradation process, the idea that autophagy regulates NMJ growth primarily through its effects on Hiw levels seems difficult to understand at first. However, recent studies demonstrate that autophagy can also operate in a substrate-selective mode in regulating specific developmental events (Rowland, 2006; Zhang, 2009). For example, in Caenorhabditis elegans, when postsynaptic cells fail to receive presynaptic contact, GABAA receptors selectively traffic to autophagosomes (Rowland, 2006). However, the detailed mechanism of such selectivity is unknown. Zhang identified SEPA-1 as a bridge that mediates the specific recognition and degradation of P granules by autophagy in C. elegans. Thus, one possibility is that Hiw is specifically targeted to autophagosomes via a mechanism that remains to be elucidated. It is also possible that many presynaptic proteins besides Hiw are degraded by autophagy, but it is the reduction in Hiw that primarily affects NMJ size. Moreover, although the idea that autophagy regulates Hiw directly is favored, the possibility cannot be ruled out that autophagy promotes degradation of Hiw through an indirect mechanism involving the proteasome or other pathway (Shen, 2009).
In principle, autophagy could be acting on either side of the NMJ to regulate its development. Because atg1+ overexpression in muscle results in lethality at the first larval instar, it was not possible to assess whether this affects NMJ growth. Although a postsynaptic role of autophagy in NMJ development cannot be ruled out, several results suggest that the effects of autophagy are primarily presynaptic: neuronal expression of UAS-atg1+ is sufficient to completely rescue the NMJ undergrowth in atg1 mutants, the Hiw-Wnd pathway functions presynaptically (Wu, 2005; Collins, 2006), and hiw is completely epistatic to autophagy for NMJ growth (Shen, 2009).
Autophagy is of particular interest as a regulator of synaptic growth because it is triggered in response to many environmental cues. These results demonstrate that decreasing or increasing autophagy from basal levels results in corresponding effects on synaptic size. Thus, autophagy is perfectly positioned to link environmental conditions with synaptic growth and plasticity. As such, it is intriguing to speculate on a role for autophagy in learning and memory (Shen, 2009).
Search PubMed for articles about Drosophila atg1
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date revised: 22 September 2021
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