InteractiveFly: GeneBrief

Autophagy-related 8a: Biological Overview | References


Gene name - Autophagy-related 8a

Synonyms -

Cytological map position - 9E6-9E7

Function - ubiquitin-like protein

Keywords - regulates autophagy in the midgut and eye - conjugated to phosphatidylethanolamine - this lipid modification anchors Atg8a into the membrane of forming and completed autophagosomes - involved in autophagosome formation, maintaining neuromuscular function and normal lifespan - mediates activation of cap-n-collar resulting in protein homeostasis

Symbol - Atg8a

FlyBase ID: FBgn0052672

Genetic map position - chrX:10,764,878-10,768,338

NCBI classification - ubiquitin-like fold

Cellular location - cytoplasmic



NCBI links: EntrezGene, Nucleotide, Protein
BIOLOGICAL OVERVIEW

A decline in protein homeostasis (proteostasis) has been proposed as a hallmark of aging. Somatic stem cells (SCs) uniquely maintain their proteostatic capacity through mechanisms that remain incompletely understood. This study describes and characterizes a 'proteostatic checkpoint' in Drosophila intestinal SCs (ISCs). Following a breakdown of proteostasis, ISCs coordinate cell cycle arrest with protein aggregate clearance by Atg8-mediated activation of the Nrf2-like transcription factor cap-n-collar C (CncC). CncC induces the cell cycle inhibitor Dacapo and proteolytic genes. The capacity to engage this checkpoint is lost in ISCs from aging flies, and it can be restored by treating flies with an Nrf2 activator, or by over-expression of CncC or Atg8a. This limits age-related intestinal barrier dysfunction and can result in lifespan extension. These findings identify a new mechanism by which somatic SCs preserve proteostasis, and highlight potential intervention strategies to maintain regenerative homeostasis (Rodriguez-Fernandez, 2019).

Protein Homeostasis (Proteostasis) encompasses the balance between protein synthesis, folding, re-folding and degradation, and is essential for the long-term preservation of cell and tissue function. It is achieved and regulated by a network of biological pathways that coordinate protein synthesis with degradation and cellular folding capacity in changing environmental conditions. This balance is perturbed in aging systems, likely as a consequence of elevated oxidative and metabolic stress, changes in protein turnover rates, decline in the protein degradation machinery, and changes in proteostatic control mechanisms. The resulting accumulation of misfolded and aggregated proteins is widely observed in aging tissues, and is characteristic of age-related diseases like Alzheimer's and Parkinson's disease. The age-related decline in proteostasis is especially pertinent in long-lived differentiated cells, which have to balance the turnover and production of long-lived aggregation-prone proteins over a timespan of years or decades. But it also affects the biology of somatic stem cells (SCs), whose unique quality-control mechanisms to preserve proteostasis are important for stemness and pluripotency (Rodriguez-Fernandez, 2019).

Common mechanisms to surveil, protect from, and respond to proteotoxic stress are the heat shock response (HSR) and the organelle-specific unfolded protein response (UPR). When activated, both stress pathways lead to the upregulation of molecular chaperones that are critical for the refolding of damaged proteins and for avoiding the accumulation of toxic aggregates. If changes to the proteome are irreversible, misfolded proteins are degraded by the proteasome or by autophagy. While all cells are capable of activating these stress response pathways, SCs deal with proteotoxic stress in a specific and state-dependent manner. Embryonic SCs (ESCs) exhibit a unique pattern of chaperone expression and elevated 19S proteasome activity, characteristics that decline upon differentiation. ESCs share elevated expression of specific chaperones (e.g., HspA5, HspA8) and co-chaperones (e.g., Hop) with mesenchymal SCs (MSCs) and neuronal SCs (NSCs), and elevated macroautophagy (hereafter referred to as autophagy) with hematopoietic SCs (HSCs), MSCs, dermal, and epidermal SCs. Defective autophagy contributes to HSC aging. It has further been proposed that SCs can resolve proteostatic stress by asymmetric segregation of damaged proteins, a concept first described in yeast (Rodriguez-Fernandez, 2019).

While these studies reveal unique proteostatic capacity and regulation in SCs, how the proteostatic machinery is linked to SC activity and regenerative capacity, and how specific proteostatic mechanisms in somatic SCs ensure that tissue homeostasis is preserved in the long term, remains to be established. Drosophila intestinal stem cells (ISCs) are an excellent model system to address these questions. ISCs constitute the vast majority of mitotically competent cells in the intestinal epithelium of the fly, regenerating all differentiated cell types in response to tissue damage. Advances made by numerous groups have uncovered many of the signaling pathways regulating ISC proliferation and self-renewal. In aging flies, the intestinal epithelium becomes dysfunctional, exhibiting hyperplasia and mis-differentiation of ISCs and daughter cells. This age-related loss of homeostasis is associated with inflammatory conditions that are characterized by commensal dysbiosis, chronic innate immune activation, and increased oxidative stress. It further seems to be associated with a loss of proteostatic capacity in ISCs, as illustrated by the constitutive activation of the unfolded protein response of the endoplasmic reticulum (UPR-ER), which results in elevated oxidative stress, and constitutive activation of JNK and PERK kinases. Accordingly, reducing PERK expression in ISCs is sufficient to promote homeostasis and extend lifespan (Rodriguez-Fernandez, 2019).

ISCs of old flies also exhibit chronic inactivation of the Nrf2 homologue CncC (Hochmuth, 2011). CncC and Nrf2 are considered master regulators of the antioxidant response, and are negatively regulated by the ubiquitin ligase Keap1. In both flies and mice, this pathway controls SC proliferation and epithelial homeostasis. It is regulated in a complex and cell-type specific manner. Canonically, Nrf2 dissociates from Keap1 in response to oxidative stress and accumulates in the nucleus, inducing the expression of antioxidant genes. Drosophila ISCs, in turn, exhibit a 'reverse stress response' that results in CncC inactivation in response to oxidative stress. This response is required for stress-induced ISC proliferation, including in response to excessive ER stress, and is likely mediated by a JNK/Fos/Keap1 pathway (Rodriguez-Fernandez, 2019).

The Nrf2 pathway has also been linked to proteostatic control: 'Non-canonical' activation of Nrf2 by proteostatic stress as a consequence of an association between Keap1 and the autophagy scaffold protein p62 has been described in mammals. A similar non-canonical activation of Nrf2 has been described in Drosophila, where CncC activation is a consequence of the interaction of Keap1 with Atg8a, the fly homologue of the autophagy protein LC3. Nrf2/CncC activation induces proteostatic gene expression, including of p62 in mammalian cells and of p62/Ref2P and LC3/Atg8a in flies. Nrf2 is further a central transcriptional regulator of the proteasome in both Drosophila and mammals. Whether and how Nrf2 also influences proteostatic gene expression in somatic SCs remains unclear (Rodriguez-Fernandez, 2019).

This study shows that Drosophila CncC links cell cycle control with proteostatic responses in ISCs via the accumulation of dacapo, a p21 cell cycle inhibitor homologue, as well as the transcriptional activation of genes encoding proteases and proteasome subunits. This study establish that this program constitutes a transient 'proteostatic checkpoint', which allows clearance of protein aggregates before cell cycle activity is resumed. In old flies, this checkpoint is impaired and can be reactivated with a CncC activator (Rodriguez-Fernandez, 2019).

The central role of Nrf2/CncC in the proteostatic checkpoint is consistent with its previously described and evolutionarily conserved influence on longevity and tissue homeostasis, and is likely to be conserved in mammalian SC populations, as Nrf2 has for example been shown to influence proliferative activity, self-renewal and differentiation in tracheal basal cells. It may be unique to somatic SCs, however, as CncC or Nrf2-mediated inhibition of cell proliferation is not observed during development (such as in imaginal discs) or in other dividing cells. Assessing the existence of an Nrf2-induced proteostatic checkpoint in mammalian SC populations will be an important future endeavor (Rodriguez-Fernandez, 2019).

Mechanistically, the results support a model in which the presence of protein aggregates activates CncC through Atg8a-mediated sequestration of Keap1. In mammals, Nrf2 activation can also be achieved through the interaction of Keap1 with the Atg8a homologue LC3 and p62, and ref2p/p62 contributes to the degradation of polyQ aggregates in Drosophila, suggesting that a conserved Atg8a/p62/Keap1 interaction may be involved in the activation of the proteostatic checkpoint. The activation of CncC after cytosolic proteostatic stress described in this study thus differs mechanistically and in its consequence from the regulation of CncC after other types of protein stress in ISCs: in response to unfolded protein stress in the ER, CncC is specifically inactivated by a ROS/JNK-mediated signaling pathway. This mechanism allows ISC proliferation to be increased in response to tissue damage, but can also contribute to the loss of tissue homeostasis in aging conditions. The activation of CncC after cytosolic protein stress, in turn, allows arresting ISC proliferation during protein aggregate clearance. The distinct responses of ISCs to cytosolic or ER-localized proteostatic stress has interesting implications for understanding of the maintenance of tissue homeostasis. While the XBP1-mediated UPR-ER allows the expansion of the ER and the induction of ER chaperones to deal with a high load of unfolded proteins in the ER, it also stimulates ISC proliferation through oxidative stress and the activation of PERK and JNK. It is tempting to speculate that the sequestration of unfolded proteins within the ER allows ISCs to proceed through mitosis without the possibility of major misregulation, while the presence of cytosolic protein aggregates may be a unique danger to the viability of the cell and its daughters. It seems likely that constitutive activation of autophagy and proteasome pathways during the clearance of cytosolic aggregates is incompatible with the need for intricate regulation of these same pathways during the cell cycle in proliferating ISCs. It will be of interest to explore this hypothesis further in the future (Rodriguez-Fernandez, 2019).

The data suggest that the coordination of cell cycle arrest and aggregate clearance is achieved by the simultaneous induction of the cell cycle inhibitor Dacapo and a battery of genes encoding proteins involved in proteolysis. While it was possible to detect dacapo transcript expression in ISCs by fluorescent in situ hybridization at 24h after HttQ138 expression, it remains unclear whether Dacapo is induced directly by CncC or via the action of a CncC target gene. It is surprising that transcriptional induction of autophagy genes in was not seen in a RNAseq experiment, but it is possible that this is due to the fact that only one timepoint was sampled after induction of protein aggregates. Since the transcriptional response of autophagy genes is likely very dynamic, a more time-resolved transcriptome analysis during aggregate formation and clearance may have captured such a response (Rodriguez-Fernandez, 2019).

It is further notable that dap deficient ISC clones exhibit a significantly higher aggregate load in these experiments than wild-type ISC clones. This suggests that the induction of proteolytic genes and of cell cycle regulators is not only coincidentally linked by CncC, but that aggregate clearance and the cell cycle arrest mediated by Dacapo need to be tightly coordinated for effective ISC proteostasis. It will be interesting to explore the mechanism of this requirement in the future. It is tempting to speculate that, as the elimination of protein aggregates requires an increase in proteasome activity, and proteasome activity can influence cell cycle timing, cell cycle inhibition is a critical safeguard against de-regulation of normal cell cycle progression (Rodriguez-Fernandez, 2019).

The data suggest that Atg8a induction in ISCs experiencing proteostatic stress may serve a dual purpose: sustained activation of the proteostatic checkpoint as well as increased autophagy flux. This dual role is distinct from other autophagy components like Atg1, since Atg1 over-expression, an efficient way of promoting autophagy in Drosophila cells, counteracts the checkpoint rather than promoting it. Exploring the relative kinetics of Atg8a and Atg1 induction in ISCs after proteostatic stress is likely to provide deeper mechanistic insight into the regulation of the proteostatic checkpoint (Rodriguez-Fernandez, 2019).

Critically, the proteostatic checkpoint is reversible. Based on the current data and previous studies, it is proposed that upon clearance of aggregates, the Keap1/Atg8a interaction is decreased, thus releasing Keap1 to inhibit CncC. Lineage-tracing studies show that this allows re-activation of ISC proliferation and recovery of normal regenerative responses (Rodriguez-Fernandez, 2019).

The loss of proteostatic checkpoint efficiency in ISCs of old guts is likely a consequence of the age-related inactivation of CncC in these cells (possibly caused by chronic oxidative stres. Accordingly, reactivating Nrf2/CncC in the gut by overexpressing CncC is sufficient to restore epithelial homeostasis in the intestine of old flies, and this study found that exposing animals to Otipraz intermittently late in life promotes epithelial barrier function and extends lifespan (Rodriguez-Fernandez, 2019).

Since Nrf2/CncC and other components required for the proteostatic checkpoint are conserved across species, it is anticipated that the current findings will be relevant to homeostatic preservation of adult SCs in vertebrates. Supporting this view, mammalian Cdkn1a (p21) has been described as an Nrf2 target gene. Transient activation of Nrf2 may thus be a viable intervention strategy to improve proteostasis and maintain regenerative capacity in high-turnover tissues of aging individuals (Rodriguez-Fernandez, 2019).

Developmentally regulated autophagy is required for eye formation in Drosophila

This study demonstrates that autophagy, an evolutionarily conserved self-degradation process of eukaryotic cells, is essential for eye development in Drosophila. Autophagic structures accumulate in a specific pattern in the developing eye disc, predominantly in the morphogenetic furrow (MF) and differentiation zone. Silencing of several autophagy genes (Atg) in the eye primordium severely affects the morphology of the adult eye through triggering ectopic cell death. In Atg mutant genetic backgrounds however genetic compensatory mechanisms largely rescue autophagic activity in, and thereby normal morphogenesis of, this organ. The results also show that in the eye disc the expression of a key autophagy gene, Atg8a, is controlled in a complex manner by the anterior Hox paralog lab (labial), a master regulator of early development. Atg8a transcription is repressed in front of, while activated along, the MF by labial. The amount of autophagic structures then remains elevated behind the moving MF. These results indicate that eye development in Drosophila depends on the cell death-suppressing and differentiating effects of the autophagic process. This novel, developmentally regulated function of autophagy in the morphogenesis of the compound eye may shed light on a more fundamental role for cellular self-digestion in differentiation and organ formation than previously thought (Billes, 2018).

Characterization of the Autophagy related gene-8a (Atg8a) promoter in Drosophila melanogaster

Autophagy is an evolutionarily conserved process which is upregulated under various stress conditions, including nutrient stress and oxidative stress. Amongst autophagy related genes (Atgs), Atg8a (LC3 in mammals) is induced several-fold during nutrient limitation in Drosophila. The minimal Atg8a cis-regulatory module (CRM) which mediates transcriptional upregulation under various stress conditions is not known. This study describes the generation and analyses of a series of Atg8a promoter deletions which drive the expression of an mCherry-Atg8a fusion cassette. Expression studies revealed that a 200 bp region of Atg8a is sufficient to drive expression of Atg8a in nutrient rich conditions in fat body and ovaries, as well as under nutrient deficient conditions in the fat body. Furthermore, this 200 bp region can mediate Atg8a upregulation during developmental histolysis of the larval fat body and under oxidative stress conditions induced by H2O2. Finally, the expression levels of Atg8a from this promoter are sufficient to rescue the lethality of the Atg8a mutant. The 200 bp promoter-fusion reporter provides a valuable tool which can be used in genetic screens to identify transcriptional and post-transcriptional regulators of Atg8a (Bali, 2017).

Epigenetic regulation of starvation-induced autophagy in Drosophila by histone methyltransferase G9a

Epigenetics is now emerging as a key regulation in response to various stresses. This study identified the Drosophila histone methyltransferase G9a (dG9a) as a key factor to acquire tolerance to starvation stress. The depletion of dG9a led to high sensitivity to starvation stress in adult flies, while its overexpression induced starvation stress resistance. The catalytic domain of dG9a was not required for starvation stress resistance. dG9a plays no apparent role in tolerance to other stresses including heat and oxidative stresses. Metabolomic approaches were applied to investigate global changes in the metabolome due to the loss of dG9a during starvation stress. The results obtained indicated that dG9a plays an important role in maintaining energy reservoirs including amino acid, trehalose, glycogen, and triacylglycerol levels during starvation. Further investigations on the underlying mechanisms showed that the depletion of dG9a repressed starvation-induced autophagy by controlling the expression level of Atg8a, a critical gene for the progression of autophagy, in a different manner to that in cancer cells. These results indicate a positive role for dG9a in starvation-induced autophagy (An, 2017).

Previous studies revealed that G9a is important for early embryogenesis and essential for viability in mice. G9a is also highly conserved among various metazoans including Drosophila, frogs (Xenopus tropicalis), fish (Danio rerio, Tetraodon nigroviridis, and Takifugu rubripes), and mammals. In Drosophila, although G9a is not essential for viability, the results of the present study suggest that G9a is conserved from the fly to mammals because of its importance in starvation stress tolerance, to which organisms are often exposed in the wild. This is also the first indication that epigenetic regulator-like G9a plays an essential role in the acquisition of starvation tolerance (An, 2017).

In order to clarify the underlying mechanisms by which the dG9a null mutant is more susceptible to starvation stress, 'bottom up' approaches have been used. Non-targeted GC-MS-based and targeted LC-MS/MS-based metabolic profiling was performed to investigate changes in the metabolome due to the loss of dG9a. The results obtained from metabolic profiles showed that dG9a played important roles in maintaining energy homeostasis, the key factor for nutrient stress tolerance. dG9a modulated energy reservoirs including amino acid, trehalose, glycogen, and TAG levels during starvation via the autophagic process. One of the unique features of the adult dG9aRG5 mutant is its higher content of glycogen under non-starved normal conditions than that of the wild-type. A previous study reported that the deletion of G9a in mouse adipose tissues promotes adipogenesis and increases body weight (Wang, 2013). These findings and the present results suggest that dG9a is also responsible for the suppression of adipogenesis, similar to mammalian G9a. Further analyses are needed in order to clarify this point (An, 2017).

The results of the present study also indicated that dG9a controlled starvation-induced autophagy by activating the expression of Atg8a; however, dG9a generally represses gene expression by dimethylating H3K9. Previous studies reported that histone and non-histone protein methylation by G9a either activated or inhibited gene expression. This study also found that the catalytic activity of dG9a was not required for the acquisition of starvation stress resistance by dG9a. This is consistent with the results of immunostaining showing that H3K9me2 levels in the nuclei of fat body cells under starvation were not significantly affected by the loss of dG9a. G9a has also been reported to activate gene expression as a molecular scaffold for the assembly of transcriptional co-activators, and the catalytic domain of G9a is not required for this function (Bittencourt, 2012). Further studies are needed in order to clarify the mechanisms by which dG9a regulates the expression of Atg8a (An, 2017).

Similar Atg8a mRNA levels were observed after 6h of fasting between wild-type and dG9aRG5 mutant flies; however, Atg8a immunostaining signals was weaker in the dG9aRG5 mutant than in the wild-type . Therefore, the loss of dG9a may repress the expression of genes that control Atg8a protein stability. Further studies are needed in order to elucidate the underlying mechanisms. During the development of Drosophila, metamorphosis is also a process that flies use to tolerate starvation stress. Even though this study demonstrated that dG9a is important for starvation stress tolerance, the viability of the dG9aRG5 mutant was not significantly less than that of the wild-type during the pupal stage. Together with the current results showing that the viability of the dG9aRG5 mutant at the larval stage was not affected by fasting conditions, the function of dG9a for starvation stress appears to be specific to the adult stage. Since programmed autophagy during the 3rd instar larval and pupal stages is well-known to be regulated by ecdysone through the PI3K pathway, starvation-induced autophagy by dG9a in the adult stage may be operated by other pathways (An, 2017).

G9a is suggested to play a positive role in the promotion of tumorigenesis in various human cancer cells such as prostate, leukemia, lung, breast, and aggressive ovarian carcinoma. The inhibition of G9a activity in cancer cells significantly inhibited cell proliferation by triggering cell cycle arrest, inducing apoptosis, or activating autophagic cell death. The novel results obtained in this study on the role of dG9a to acquire starvation tolerance may also make it possible to explain the positive role of G9a in the promotion of tumorigenesis. Cells inside a tumor mass are exposed to starvation conditions because nutrients are not fully supplied to these cells. In order to overcome starvation stress, autophagy is induced in these cells. Therefore, G9a may play a role in the acquisition of starvation tolerance in cells in the tumor mass. The present study found that the loss of dG9a led to the inactivation of starvation-induced autophagy due to a decrease in Atg8a levels. In contrast, a previous study on cancer cells showed that the loss of G9a during starvation activated the transcription of LC3B (the Atg8a ortholog in mammals) and triggered autophagy. Taken together, these results suggest that the epigenetic gene regulation of G9a depends on cell/tissue types (An, 2017).

Glial Draper rescues Abeta toxicity in a Drosophila model of Alzheimer's disease

Pathological hallmarks of Alzheimer's disease (AD) include amyloid-beta (Abeta) plaques, neurofibrillary tangles, and reactive gliosis. Glial cells offer protection against AD by engulfing extracellular Abeta peptides, but the repertoire of molecules required for glial recognition and destruction of Abeta are still unclear. This study shows that the highly conserved glial engulfment receptor Draper/MEGF10 provides neuroprotection in an AD model of Drosophila (both sexes). Neuronal expression of human Abeta42arc in adult flies results in robust Abeta accumulation, neurodegeneration, locomotor dysfunction, and reduced lifespan. Notably, all of these phenotypes are more severe in draper mutant animals, while enhanced expression of glial Draper reverses Abeta accumulation, as well as behavioral phenotypes. Stat92E, c-Jun N-terminal Kinase (JNK)/AP-1 signaling, and expression of matrix metalloproteinase-1 (Mmp1) are activated downstream of Draper in glia in response to Abeta42arc exposure. Furthermore, Abeta42-induced upregulation of the phagolysosomal markers Atg8 and p62 was notably reduced in draper mutant flies. Based on these findings, it is proposed that glia clear neurotoxic Abeta peptides in the AD model Drosophila brain through a Draper/STAT92E/JNK cascade that may be coupled to protein degradation pathways such as autophagy or more traditional phagolysosomal destruction methods (Ray, 2017).

p62/sequestosome-1, Autophagy-related Gene 8, and autophagy in Drosophila are regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), independent of transcription factor TFEB

The selective autophagy receptor p62/sequestosome 1 (SQSTM1) interacts directly with LC3 and is involved in oxidative stress signaling in two ways in mammals. First, p62 is transcriptionally induced upon oxidative stress by the NF-E2-related factor 2 (NRF2) by direct binding to an antioxidant response element (ARE) in the p62 promoter. Secondly, p62 accumulation, occurring when autophagy is impaired, lead to increased p62 binding to the NRF2 inhibitor KEAP1 resulting in reduced proteasomal turnover of NRF2. This gives chronic oxidative stress signaling through a feed forward loop. This study shows that the Drosophila p62/SQSTM1 orthologue, Ref(2)P, interacts directly with Atg8a via a LC3-interacting region (LIR) motif, supporting a role for Ref(2)P in selective autophagy. The ref(2)P promoter also contains a functional ARE that is directly bound by the NRF2 orthologue, CncC which can induce ref(2)P expression along with the oxidative stress associated gene gstD1. However, distinct from the situation in mammals, Ref(2)P does not interact directly with DmKeap1 via a KEAP1-interacting region (KIR) motif. Neither does ectopically expressed Ref(2)P, nor autophagy deficiency, activate the oxidative stress response. Instead, DmAtg8a interacts directly with DmKeap1, and DmKeap1 is removed upon programmed autophagy in Drosophila gut cells. Strikingly, CncC induced increased Atg8a levels and autophagy independent of TFEB/MitF in fat body and larval gut tissues. Thus, these results extend the intimate relationship between oxidative stress sensing NRF2/CncC transcription factors and autophagy, and suggests that NRF2/CncC may regulate autophagic activity in other organisms too (Jain, 2015).

Autophagy is a catabolic process where an isolation membrane engulfs part of the cytoplasm to create a double-membrane vesicle called the autophagosome, which fuses with lysosomes and leads to degradation of their contents. Selective autophagy receptors bind to cargo and dock onto the forming phagophore through a direct interaction with ATG8 family proteins, enabling delivery and autophagic degradation of the cargo. Human p62/sequestosome 1 (hereafter named p62) interacts with LC3 and ubiquitin, is a selective autophagic substrate, and is the first identified cargo receptor for autophagic degradation of ubiquitinated targets. When autophagy is abolished in the liver of Atg7 conditional knock-out mice, p62 accumulates in aggregates, and antioxidant proteins and phase II detoxification enzymes are strongly induced. p62 is induced by various stressors both at the mRNA and protein levels, and this p62 induction is inhibited in cells from Nrf2 knock-out mice. Several groups have reported that p62 competes with NRF2 for binding to KEAP1, resulting in stabilization of NRF2, whereas KEAP1 is sequestered into p62 bodies and subsequently degraded by autophagy. It was also recently shown that phosphorylation of the KEAP1-interacting region (KIR) motif of p62 enhanced binding to KEAP1. It has been reported earlier that NRF2 bound to an ARE site in the p62 promoter and induced p62 expression upon oxidative stress. Hence, it was not possible to conclude that p62 is involved in establishing a positive feedback loop inducing its own expression and prolonged NRF2 response under stress conditions (Jain, 2015).

D. melanogaster ref(2)P is the orthologue of mammalian p62 and was first characterized as a modifier of σ virus multiplication. Ref(2)P has been reported to be a major component of protein aggregates in flies defective in autophagy or with impaired proteasomal function and in fly models of neurodegenerative diseases. However, it is not known if Ref(2)P binds directly to DmAtg8 via a functional LIR motif (Jain, 2015).

This study shows that Ref(2)P interacts with DmAtg8a in vitro and in vivo through a LIR motif and that this is necessary for autophagic degradation of Ref(2)P. ref(2)P is a transcriptional target of CncC and contains a CncC-responsive ARE in its promoter. However, Ref(2)P does not bind directly to DmKeap1 via a KIR motif, as found for mammalian p62 and KEAP1. Consequently, ectopically expressed Ref(2)P does not induce the oxidative stress response in fly tissues. Very interestingly, this study found CncC induces atg8a and stimulates autophagy in the fat body and larval gut. Hence, the positive feedback loop between p62 and Nrf2 seen in mammals is not present in D. melanogaster. However, CncC can induce ref(2)P, atg8a, and autophagy (Jain, 2015).

Ref(2)P, the single p62 orthologue in D. melanogaster, is an established signaling adapter (Avila, 2002; Carre-Mlouka, 2007; Moscat, 2007). Similar to p62, Ref(2)P accumulates with ubiquitin-containing protein aggregates in the brain of autophagy-deficient and neurodegenerative mutants of Drosophila. Ref(2)P is involved in maintenance of the viable mitochondria pool by acting downstream of Pink1 and Parkin, where Ref(2)P recycles excessive unfolded proteins via autophagy (Pimenta de Castro, 2012). This indicates a role for Ref(2)P as an autophagy receptor, similar to mammalian p62. Moreover, a recent report suggests that Ref(2)P is a selective autophagy substrate, but direct binding of Ref(2)P to DmAtg8a has not been shown. This study found that Ref(2)P interacts with DmAtg8a in a LC3-interacting region (LIR)-dependent manner. The Ref(2)P LIR also fulfills the requirement for a canonical LIR motif. The functional importance of the LIR motif in Ref(2)P was demonstrated by its requirement for accumulation of Ref(2)P in acidic vesicles and subsequent autophagic degradation (Jain, 2015).

Only the longest Cnc isoform, CncC, contains the Keap1-interacting DLG and ETGE motifs. Homology with the Neh2 domain of NRF2 suggests CncC to be the direct homologue of NRF2. Similar to NRF2, CncC is thought to interact with DmKeap1 (Kobayashi, 2002), and the activity of CncC is inhibited by DmKeap1 (Chatterjee, 2012). This study tested all three isoforms of Cnc (A, B, and C) for binding to DmKeap1, and only CncC interacted. The binding is mediated by the conserved ETGE motif. It has been reported both in humans and rodents that p62 binds directly to Keap1 using an ETGE-like motif, and this interaction positively regulates Nrf2 by blocking the interaction between Keap1 and Nrf2. A simple protein-protein interaction map involving p62/Ref(2)P, KEAP1/DmKeap1, NRF2/CncC, and ATG8/DmAtg8a highlights the major differences and similarities between humans and D. melanogaster. Surprisingly, no direct interaction was found between Ref(2)P and DmKeap1 in Drosophila. This was supported by reporter gene assays where Ref(2)P did not activate its own promoter, as p62 does in mammals. Recently, Ref(2)P and DmKeap1 were reported to be co-immunoprecipitated from Drosophila cells. This study found this to be mediated by the UBA domain of Ref(2)P, which probably recognized ubiquitinated DmKeap1. Hence, the direct KIR-mediated interaction between p62 and Keap1 evolved with the vertebrates, consistent with the lack of a KIR motif in p62 orthologues in non-chordate metazoans (Jain, 2015).

CncC has a central role in regulation of xenobiotic response, cellular stress, and electrophilic stress. CncC significantly induced the ref(2)P promoter, CncB gave no induction, and CncA had a significant negative effect. This is most probably due to the lack of the N-terminal transactivation domain found in CncC. Overexpressed CncC is a proteasome substrate, not detectable under normal conditions, but is stabilized by inhibition or depletion of proteasome subunit S5a. Consistently, it was not possible to easily detect overexpressed CncC by Western blot in cultured cells unless proteasomal degradation was inhibited. However, CncA and CncB were very well expressed under similar conditions. This is interesting, because only CncC has the DLG and ETGE motifs for Keap1 binding. This indicates that DmKeap1 may work as an E3 ligase adaptor protein to recruit CncC for its proteasomal degradation as found for mammalian Keap1. The current data suggest that CncA and -B may compete with CncC for DNA binding to the ARE on the ref(2)P promoter. This indicates a role for the CncA and CncB isoforms as competitive repressors of CncC under non-stress conditions, to maintain homeostasis in the D. melanogaster antioxidant defense system. This is in analogy to p65 (truncated isoform of Nrf1) and a caspase-cleaved form of Nrf2, which are bothreported to act as transcriptional repressors in vertebrates (Jain, 2015).

Consistent with the results from cell culture experiments, CncC overexpression induced ref(2)P and the target gene gstD in hindgut, wing discs, and fat bodies of D. melanogaster. These in vivo results strongly support the finding that ref(2)P is a target gene for CncC. Interestingly, overexpression of Ref(2)P did not induce an oxidative stress response, at least not as measured by gstD-GFP expression in hindgut and wing discs. This correlates with the lack of a ETGE-like motif in Ref(2)P and confirms the absence of a ref(2)P-mediated positive feedback loop in D. melanogaster (Jain, 2015).

Surprisingly, this study found a direct interaction between DmKeap1 and DmAtg8a. The canonical LIR-LDS interaction is dependent on both the N-terminal part (residues 1-28) and the C-terminal part (residues 30-125) of LC3B. However, the mode of the DmKeap1-DmAtg8a interaction seems different because DmKeap1 could bind to the N-terminal 71 amino acids of DmAtg8a(1-71). It has not been possible to map any motif mediating this non-canonical interaction. However, the interaction of DmKeap1 with DmAtg8a is interesting, both because DmKeap1 appears to recognize a different binding surface in DmAtg8a than Ref(2)P and other LIR-containing proteins and because the interaction might have an important role for DmAtg8a-mediated autophagic degradation of DmKeap1 during Drosophila development. Recent studies show mammalian Keap1 to be degraded by autophagy under nutritional starvation and oxidative stress in a p62-dependent manner, whereas degradation of mammalian Keap1 by basal autophagy has not been clearly demonstrated. Consistent with this, no degradation of DmKeap1 by basal autophagy was observed in Drosophila, but DmKeap1 was degraded under programmed autophagy during Drosophila development. The significance of the DmAtg8a-DmKeap1 interaction for the degradation of DmKeap1 by autophagy remains to be tested. The idea is favored that autophagic degradation of DmKeap1 depends on a co-recruitment of autophagy receptors like Ref(2)P, and this is strongly supported by the finding that Ref(2)P interacts with ubiquitinated DmKeap1 in cell culture. Possibly, a combined binding of Ref(2)P and DmKeap1 to DmAtg8 may help to increase the local concentration of DmAtg8 to secure an efficient encapsulation of the aggregate. However, further work is needed to reveal the underlying mechanism and significance of this interaction (Jain, 2015).

The finding that there is no positive feedback loop between CncC and Ref(2)P in flies was quite unexpected. However, the introduction of a KIR motif in p62 homologs during evolution of the most primitive fish, the amphioxus, suggests that the interdependent role of p62 and NRF2 in oxidative stress regulation developed early in vertebrate evolution. A related type of positive feedback loop has been reported for mammalian p62 and NFκB, predicting a putative cross-talk between NRF2 and NFκB pathways. Both gain of function mutations in NRF2 and loss of function mutations in Keap1 have been identified in human cancers and are believed to contribute to cancer cell survival and stress resistance upon cancer treatment. This study has found that loss of Keap1 or CncC gain of function induces Atg8a up-regulation and autophagy. These results are thought provoking, because autophagy, like NRF2 gain of function, has been shown to prevent initial tumor development. Once established, however, autophagy promotes cancer cell survival during stress conditions and cancer treatment. Under which physiological settings CncC-mediated control of autophagy may function remains an open question. The most obvious possibility is that CncC controls autophagy in response to reactive oxygen species. Previous studies have established that Drosophila utilizes a TRAF6/Atg9/Jun N-terminal kinase (JNK) stress pathway to activate autophagy upon oxidative stress provoked by hydrogen peroxide feeding. In concordance with those studies, this study found no evidence that CncC activity is required for autophagy induced upon hydrogen peroxide feeding. It remains possible that more subtle and physiological conditions of reactive oxygen species formation during aging, mitochondrial dysfunction, or oncogene-induced stress may enlist CncC in stress coping mechanisms involving autophagy. It will be interesting to pursue potential roles of CncC in in vivo cancer models (Jain, 2015).

Drosophila Gyf/GRB10 interacting GYF protein is an autophagy regulator that controls neuron and muscle homeostasis

Autophagy is a process essential for eliminating ubiquitinated protein aggregates and dysfunctional organelles. Defective autophagy is associated with various degenerative diseases such as Parkinson's disease. Through a genetic screening in Drosophila, this study identified CG11148, whose product is orthologous to GIGYF1 (GRB10 interacting GYF protein 1) and GIGYF2 in mammals, as a new autophagy regulator; the gene is hereafter refered to as Gyf. Silencing of Gyf completely suppressed the effect of Atg1-Atg13 activation in stimulating autophagic flux and inducing autophagic eye degeneration. Although Gyf silencing did not affect Atg1-induced Atg13 phosphorylation or Atg6-Pi3K59F (class III PtdIns3K)-dependent Fyve puncta formation, it inhibited formation of Atg13 puncta, suggesting that Gyf controls autophagy through regulating subcellular localization of the Atg1-Atg13 complex. Gyf silencing also inhibited Atg1-Atg13-induced formation of Atg9 puncta, which is accumulated upon active membrane trafficking into autophagosomes. Gyf-null mutants also exhibited substantial defects in developmental or starvation-induced accumulation of autophagosomes and autolysosomes in the larval fat body. Furthermore, heads and thoraxes from Gyf-null adults exhibited strongly reduced expression of autophagosome-associated Atg8a-II compared to wild-type (WT) tissues. The decrease in Atg8a-II was directly correlated with an increased accumulation of ubiquitinated proteins and dysfunctional mitochondria in neuron and muscle, which together led to severe locomotor defects and early mortality. These results suggest that Gyf-mediated autophagy regulation is important for maintaining neuromuscular homeostasis and preventing degenerative pathologies of the tissues. Since human mutations in the GIGYF2 locus were reported to be associated with a type of familial Parkinson disease, the homeostatic role of Gyf-family proteins is likely to be evolutionarily conserved (Kim, 2015).

Aging and autophagic function influences the progressive decline of adult Drosophila behaviors

Middle-aged wild-type flies (WT, ~4-weeks) exhibit a marked accumulation of neural aggregates that is commensurate with the decline of the autophagy pathway. However, enhancing autophagy via neuronal over-expression of Atg8a (Atg8a-OE) reduces the age-dependent accumulation of aggregates. Only modest behavioral changes occur by 4-weeks of age, with the noted exception of group-housed male flies. Male flies in same-sex social groups exhibit a progressive increase in nighttime activity. Infrared videos show aged group-housed males (4-weeks) are engaged in extensive bouts of courtship during periods of darkness, which is partly repressed during lighted conditions. Together, these nighttime courtship behaviors are nearly absent in young WT flies and aged Atg8a-OE flies. Previous studies have indicated a regulatory role for olfaction in male courtship partner choice. Coincidently, the mRNA expression profiles of several olfactory genes decline with age in WT flies; however, they are maintained in age-matched Atg8a-OE flies. Together, these results suggest that middle-aged male flies develop impairments in olfaction, which could contribute to the dysregulation of courtship behaviors during dark time periods. Combined, these results demonstrate that as Drosophila age, they develop early behavior defects that are coordinate with protein aggregate accumulation in the nervous system. In addition, the nighttime activity behavior is preserved when neuronal autophagy is maintained (Atg8a-OE flies). Thus, environmental or genetic factors that modify autophagic capacity could have a positive impact on neuronal aging and complex behaviors (Ratliff, 2015).

Different effects of Atg2 and Atg18 mutations on Atg8a and Atg9 trafficking during starvation in Drosophila

The Atg2-Atg18 complex acts in parallel to Atg8 and regulates Atg9 recycling from phagophore assembly site (PAS) during autophagy in yeast. This study shows that in Drosophila, both Atg9 and Atg18 are required for Atg8a puncta formation, unlike Atg2. Selective autophagic degradation of ubiquitinated proteins is mediated by Ref(2)P/p62. The transmembrane protein Atg9 accumulates on refractory to Sigma P (Ref(2)P) aggregates in Atg7, Atg8a and Atg2 mutants. No accumulation of Atg9 is seen on Ref(2)P in cells lacking Atg18 or Vps34 lipid kinase function, while the Atg1 complex subunit FIP200 is recruited. The simultaneous interaction of Atg18 with both Atg9 and Ref(2)P raises the possibility that Atg18 may facilitate selective degradation of ubiquitinated protein aggregates by autophagy (Nagy, 2014).

Age-induced reduction of autophagy-related gene expression is associated with onset of Alzheimer's disease

Aging is a major risk factor for Alzheimer's disease (AD). Aggregation of amyloid β (Aβ) in cerebral cortex and hippocampus is a hallmark of AD. Many factors have been identified as causative elements for onset and progression of AD; for instance, tau seems to mediate the neuronal toxicity of Aβ, and downregulation of macroautophagy (autophagy) is thought to be a causative element of AD pathology. Expression of autophagy-related genes is reduced with age, which leads to increases in oxidative stress and aberrant protein accumulation. This study found that expression of the autophagy-related genes atg1, atg8a, and atg18 in Drosophila melanogaster was regulated with aging as well as their own activities. In addition, the level of atg18 was maintained by dfoxo (foxo) and dsir2 (sir2) activities in concert with aging. These results indicate that some autophagy-related gene expression is regulated by foxo/sir2-mediated aging processes. It was further found that reduced autophagy activity correlated with late-onset neuronal dysfunction caused by neuronal induction of Aβ. These data support the idea that age-related dysfunction of autophagy is a causative element in onset and progression of AD (Omata, 2014).

Activin signaling targeted by insulin/dFOXO regulates aging and muscle proteostasis in Drosophila

Reduced insulin/IGF signaling increases lifespan in many animals. To understand how insulin/IGF mediates lifespan in Drosophila, chromatin immunoprecipitation-sequencing analysis was performed with the insulin/IGF regulated transcription factor dFOXO in long-lived insulin/IGF signaling genotypes. Dawdle, an Activin ligand, is bound and repressed by dFOXO when reduced insulin/IGF extends lifespan. Reduced Activin signaling improves performance and protein homeostasis in muscles of aged flies. Activin signaling through the Smad binding element inhibits the transcription of Autophagy-specific gene 8a (Atg8a) within muscle, a factor controlling the rate of autophagy. Expression of Atg8a within muscle is sufficient to increase lifespan. These data reveal how insulin signaling can regulate aging through control of Activin signaling that in turn controls autophagy, representing a potentially conserved molecular basis for longevity assurance. While reduced Activin within muscle autonomously retards functional aging of this tissue, these effects in muscle also reduce secretion of insulin-like peptides at a distance from the brain. Reduced insulin secretion from the brain may subsequently reinforce longevity assurance through decreased systemic insulin/IGF signaling (Bai, 2013).

Insulin/IGF-1 signaling modulates longevity in many animals. Genetic analysis in C. elegans and Drosophila shows that insulin/IGF-1 signaling requires the DAF-16/FOXO transcription factor to extend lifespan, while in humans several polymorphisms of FoxO3A are associated with exceptional longevity. Although many downstream effectors of FOXO have been identified through genome-wide studies, the targets of FOXO responsible for longevity assurance upon reduced insulin signaling are largely unknown. This study found 273 genes targeted by Drosophila FOXO using ChIP-Seq with two long-lived insulin mutant genotypes. Focused was placed on daw, an Activin ligand, which is transcriptionally repressed by FOXO upon reduced insulin/IGF signaling. Inactivation of daw and of its downstream signaling partners babo and Smox extend lifespan. These results are reminiscent of observations from C. elegans where reduced TGF-β/dauer signaling extends longevity. Notably, the lifespan extension of TGF-β/dauer mutants (e.g. daf-7 (e1372) mutants) can be suppressed by daf-16 mutants, suggesting that TGF-β signaling intersects with the insulin/IGF-1 pathway for longevity in C. elegans. In phylogenetic analysis, DAF-7, Daw and mammalian Activin-like proteins share common ancestry. Activin signaling, in response to insulin/IGF-1, may thus represent a taxonomically conserved longevity assurance pathway (Bai, 2013).

Longevity benefits of reduced Activin (TGF-β/dauer) in C. elegans were resolved only when the matricide or 'bagging' (due to progeny hatching within the mother) was prevented by treating daf-7(e1372) mutants with 5-fluorodeoxyuridine (FUdR) to block progeny development. This approach made it possible to distinguish the role of Activin in somatic aging from the previously recognized influence of BMP (Sma/Mab signaling) upon reproductive aging in C. elegans. Activin, of course, is a somatically expressed regulatory hormone of mammalian menstrual cycles that induces follicle-stimulating hormone (FSH) in the pituitary gland. In young females, FSH is suppressed within a cycle when maturing follicles secrete the related TGF-β hormone Inhibin. In mammalian reproductive aging, the effect of Activin in the pituitary becomes unopposed as the stock of primary follicles declines, thus inducing elevated production of FSH. This study now found that reduced Activin but not BMP signaling favors somatic persistence in Drosophila. These parallels between reproductive and somatic aging among invertebrate models and humans suggest that unopposed Activin signaling is pro-aging while favoring reproduction (Bai, 2013).

Reduced insulin/IGF signaling extends lifespan through interacting autonomous and non-autonomous actions. Reducing IIS in some distal tissues has been shown to slow aging because this reduces insulin secretion from a few neurons: reducing IIS by increasing dFOXO in fat body or muscle extends Drosophila fly lifespan while decreasing IPC production of systemically secreted DILP2. This study has identified Activin as a direct, downstream target of insulin/dFOXO signaling within muscles that has the capacity to non-autonomously regulate lifespan. Knockdown of Activin in muscle but not in fat body is sufficient to prolong lifespan. RNAi for muscle Activin signaling led to decreased circulating DILP2 and increased peripheral insulin signaling. Muscle is thus proposed to produce a signaling factor, a myokine, which impacts organism-wide aging and metabolism (Bai, 2013).

Aging muscle may produce different myokine-like signals in response to their physiological state. Aged muscles degenerate in many ways including changes in composition, mitochondria, regenerative potential and within-cell protein homeostasis. Protein homeostasis is normally maintained, at least in part, by autophagy. Loss of macroautophagy and chaperone-mediated autophagy with age will accelerate the accumulation of damaged proteins. Expression of Atg8a in Drosophila CNS is reported to extend lifespan by 56% (Simonsen, 2008), while recent studies find elevated autophagy in long-lived mutants including those of the insulin/IGF-1 signaling pathway. The current results show that insulin/IGF signaling can regulate autophagy through its control of Activin via dFOXO. Poly-ubiquitinated proteins accumulate in aging Drosophila while lysosome activity and macroautophagy decline. Muscle performance with age (flight, climbing) was preserved by inactivating Activin within this tissue. This genetic treatment also reduced the accumulation of protein aggregates. These effects are mediated by blocking the transcription factor Smox, which otherwise represses Atg8a. Smox directly regulates Atg8a through its conserved Smad binding motif (AGAC AGAC). These results, however, contrast with an observation where TGF-β1 promotes autophagy in mouse mesangial cells (Bai, 2013).

Insulin/IGF-1 signaling is a widely conserved longevity assurance pathway. The data indicate that reduced insulin/IGF-1 retards aging at least in part through its FOXO-mediated control of Activin. Furthermore, affecting Activin only in muscle is sufficient to slow its functional decline as well as to extend lifespan. Autophagy within aging muscle controls these outcomes, and it is now found that Activin directly regulates autophagy through Smox-mediated repression of Atg8a. If extrapolated to mammals, pharmaceutical manipulations of Activin may reduce age-dependent muscle pathology associated with impaired autophagy, and potentially increase healthy and total lifespan through beneficial signaling derived from such preserved tissue (Bai, 2013).

Uba1 functions in Atg7- and Atg3-independent autophagy

Autophagy is a conserved process that delivers components of the cytoplasm to lysosomes for degradation. The E1 and E2 enzymes encoded by Atg7 and Atg3 are thought to be essential for autophagy involving the ubiquitin-like protein Atg8. This study describes an Atg7- and Atg3-independent autophagy pathway that facilitates programmed reduction of cell size during intestine cell death. Although multiple components of the core autophagy pathways, including Atg8, are required for autophagy and cells to shrink in the midgut of the intestine, loss of either Atg7 or Atg3 function does not influence these cellular processes. Rather, Uba1, the E1 enzyme used in ubiquitylation, is required for autophagy and reduction of cell size. These data reveal that distinct autophagy programs are used by different cells within an animal, and disclose an unappreciated role for ubiquitin activation in autophagy (Chang, 2013).

Macroautophagy (autophagy) is a system that is used to transfer cytoplasmic material, including proteins and organelles, to lysosomes by all eukaryotic cells. Autophagy is augmented during cell stress to reduce damage to enable cell survival, and is also associated with the death of animal cells. Although most studies of this process have focused on stress-induced autophagy, such as nutrient deprivation, autophagy is also a normal aspect of animal development where it is required for proper death and removal of cells and tissues. Defects in autophagy lead to accumulation of protein aggregates and damaged organelles, as well as human disorders. Most of the knowledge about the genes controlling autophagy is based on pioneering studies in the yeast Saccharomyces cerevisiae, and it is not clear whether cells that exist in extremely different contexts within multi-cellular organisms could use alternative factors to regulate this catabolic process (Chang, 2013).

Atg genes that are conserved from yeast to humans are required for autophagy, and include the Atg1 and Vps34 regulatory complexes, as well as two ubiquitin-like conjugation pathways. The two ubiquitin-like molecules, named Atg8 (LC3 and GABARAP in mammals) and Atg12, become associated with the isolation membranes that form autophagosomes through the activity of the E1 enzyme Atg7. Atg3 functions as the E2-conjugating enzyme for Atg8, and Atg10 functions as the E2 for Atg12. Atg12 associates with Atg5 and Atg16 during the formation of the autophagosome, and Atg8 is conjugated to the lipid phosphatidyl-ethanolamine enabling this protein to associate with the isolation membrane and autophagosome. Lipidated Atg8 remains associated with autophagosomes until fusion with lysosomes to form autolysosomes where cargoes are degraded by lysosomal enzymes (Chang, 2013).

Degradation of the midgut of the Drosophila melanogaster intestine involves a large change in midgut length, has elevated autophagy and markers of caspases associated with it, requires autophagy, and seems to be caspase independent (Denton, 2009, Lee, 2002; Micchelli, 2011). This study shows that autophagy is required for programmed reduction in cell size at the onset of intestine cell death in Drosophila. Atg genes encoding components of the Atg1 and Vps34 complexes are required for midgut cell autophagy and reduction in size. Surprisingly, although Atg8a is required for autophagy and programmed cell size reduction, the evolutionarily conserved E1-activating enzyme Atg7 and E2-conjugating enzyme Atg3 are not required for these cellular events. This study screened the E1-activating enzymes encoded by the fly genome and identified Uba1 as being required for autophagy and reduction of cell size during midgut cell death. Although the genes that control autophagy are conserved throughout eukaryotes, the current data provide evidence indicating that the core autophagy machinery may not be identical in all cells within an organism (Chang, 2013).

Autophagy has been shown to influence cell size during growth factor and nutrient restriction in mammalian cells lines, but this study indicates that autophagy controls cell size as part of a normal developmental program. The discovery that Atg7 and Atg3 are not required for autophagy and cell size reduction in dying midgut cells in Drosophila is surprising. Although an Atg5, Atg7- and LC3-independent autophagy pathway has been reported (Nishida, 2009), this study describes autophagy that requires Atg8 (LC3) and does not require Atg7 and Atg3. It has been assumed that components of the core Atg8 (LC3) and Atg12 conjugation pathways are used by all eukaryotic cells, but this study provides evidence that alternative factors can function to regulate autophagy in a cell-context-specific manner (Chang, 2013).

This study highlights that autophagy may have different regulatory mechanisms in distinct cell types within an animal. Different forms of autophagy could involve either unique regulatory pathways , different amounts and rates of autophagy or alternative cargo selection mechanisms, and these are not mutually exclusive. Another possibility is that differences in cargo selection alone, perhaps based on specific cargo adaptor proteins, could mediate a distinct type of autophagy (Chang, 2013).

This paper reports that an E1 enzyme other than Atg7 is required for Atg8 and Atg5 puncta formation, and clearance of ubiquitin-binding protein p62 and mitochondria. The studies indicate that Uba1 fails to function in place of Atg7, as expected on the basis of the unique architecture and use of ubiquitin-like proteins and E2-binding domains in these highly divergent E1 enzymes. Although the possibility cannot be excluded that Atg8a is activated by unknown factors, the simplest model to explain the data positions Uba1 function at a different stage of the autophagy process that depends on ubiquitin conjugation. Previous work in a mammalian cell line indicated that Uba1 is required for protein degradation by lysosomes, but this was not because of decreased autophagosome formation. In addition, recent work in Drosophila implicated the de-ubiquitylation enzyme USP36 in autophagy. However, the inability of Atg5 knockdown to suppress the USP36 mutant phenotype, as well as the accumulation of both GFP-Atg8a and ubiquitin-binding protein p62 in USP36 mutant cells, suggests a defect in autophagic flux rather than a defect in the formation of autophagosomes. p62 and other ubiquitin-binding proteins are known to facilitate recruitment of ubiquitylated cargoes into autophagosomes (Johansen, 2011). In addition, p62 was recently shown to accumulate at sites of autophagosome formation even when autophagosome formation is blocked. Thus, it is possible that Uba1 promotes cargo recruitment to the sites of autophagosome formation to facilitate autophagy. However, it is also possible that Uba1 could function at multiple stages in the regulation of autophagy (Chang, 2013).

It is critical to understand the mechanisms that regulate autophagy given the interest in this catabolic process as a therapeutic target for multiple age-associated disorders, including cancer and neurodegeneration. Significantly, these studies illuminate that autophagy has different regulatory mechanisms in distinct cell types within an animal, and highlight the importance of studying core autophagy genes in specific cell types under physiological conditions (Chang, 2013).

Promoting basal levels of autophagy in the nervous system enhances longevity and oxidant resistance in adult Drosophila

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 demonstrated 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).

The PI 3-kinase regulator Vps15 is required for autophagic clearance of protein aggregates

Autophagy is involved in cellular clearance of aggregate-prone proteins, thereby having a cytoprotective function. Studies in yeast have shown that the PI 3-kinase Vps34 and its regulatory protein kinase Vps15 are important for autophagy, but the possible involvement of these proteins in autophagy in a multicellular animal has not been addressed genetically. This study created a Drosophila deletion mutant of vps15 and investigated its role in autophagy and aggregate clearance. Homozygous δvps15 Drosophila died at the early L3 larval stage. Using GFP-Atg8a as an autophagic marker, fluorescence microscopy was employed to demonstrate that fat bodies of wild type Drosophila larvae accumulated autophagic structures upon starvation whereas δvps15 fat bodies showed no such response. Likewise, electron microscopy revealed starvation-induced autophagy in gut cells from wild type but not δvps15 larvae. Fluorescence microscopy showed that δvps15 mutant tissues accumulated profiles that were positive for ubiquitin and Ref(2)P, the Drosophila homolog of the sequestosome marker SQSTM1/p62. Biochemical fractionation and Western blotting showed that these structures were partially detergent insoluble, and immuno-electron microscopy further demonstrated the presence of Ref(2)P positive membrane free protein aggregates. These results provide the first genetic evidence for a function of Vps15 in autophagy in multicellular organisms and suggest that the Vps15- containing PI 3-kinase complex may play an important role in clearance of protein aggregates (Lindmo, 2008).

Studies of the involvement of specific PI3Ks in autophagy in higher organisms such as Drosophila and mammals by pharmacological PI3K inhibitors have been complicated by the fact that these animals express multiple classes of PI3Ks that may have opposing roles. It has been found that class I PI3K represses autophagy during the early larval stages in Drosophila, and that its downregulation in response to ecdysone signaling triggers developmental autophagy. The present study sought to clarify the possible involvement of class III PI3K in autophagy and aggregate clearance by generating a Drosophila mutant in which the gene for the regulatory Vps15 subunit was deleted. The δvps15 mutant larvae turned out to be defective for starvation induced autophagy. Importantly, vps15 mutant animals accumulated detergent-soluble and -insoluble structures that are likely to represent endosomes and sequestosomes, respectively. This provides evidence for the involvement of Vps15 in autophagy and aggregate clearance in metazoans (Lindmo, 2008).

The only PI3K in S. cerevisae, Vps34, can participate in two distinct protein complexes; one consisting of Vps34, Vps15, Vps30 and Vps38 that functions in vacuolar protein sorting and one consisting of Vps34, Vps15, Vps30 and Atg14 that functions in autophagy (Kihara, 2001). So far, no metazoan homolog of Atg14 has been reported, whereas metazoan homologs of Vps34, Vps15 and Vps30 are known. Of these, the Vps30 homolog, Beclin-1, an interactor of the antiapoptotic proteins Bcl-2 and Bcl-XL, has been most studied for its role in autophagy in metazoans. Overexpression of Beclin-1 in MCF7 breast carcinoma cells promotes autophagy and inhibits cell proliferation, whereas its depletion promotes apoptosis. The possible role of Beclin-1 in aggregate clearance has not been investigated, nor have metazoan Vps34 and Vps15 been studied in this context. It was therefore considered important to study whether the metazoan Vps34-Vps15 subcomplex is required for autophagy and aggregate clearance. Because of the availability of appropriate Drosophila FRT strains, a specific deletion of the vps15 gene was generated in Drosophila. The inhibition of starvation-induced autophagy in gut and fat body tissues of δvps15 larvae demonstrates the importance of the Vps15 for autophagy in metazoans. Most importantly, the accumulation of protein aggregates in the δvps15 mutants shows that this complex is critically required for normal clearance of such aggregates (Lindmo, 2008).

The polyubiquitin binding p62 protein accumulates strongly on ubiquitin-positive protein aggregates and serves as a reporter for such structures. Protein aggregates are not formed in p62/Ref(2)P mutants and the fact that p62 binds directly to the mammalian Atg8 homolog LC3 and recruits it to ubiquitin-positive aggregates suggests that p62 may serve to mark the protein aggregates for autophagic degradation. The present report used antibodies against conjugated ubiquitin and the Drosophila homolog of p62, Ref(2)P, as a marker for protein aggregates. Although Ref(2)P was originally identified as a factor involved in male fertility and sigma virus replication, it contains all the structural hallmarks of a p62 ortholog, including the PB1, ZZ and UBA domains. Interestingly, δvps15 Drosophila larvae accumulated numerous Ref(2)P-positive structures, indicative of impaired metabolism of protein aggregates. Consistent with this, the δvps15 mutants also accumulated ubiquitin-positive structures. Because depletion of certain proteins involved in endocytic trafficking causes the accumulation of ubiquitinated membrane proteins in early endosomes, some of the ubiquitin- and Ref(2)P positive profiles might correspond to endosomes. This is supported by the finding that a fraction of the ubiquitin- and Ref(2)P-positive structures could be solubilized in Tx. Because confocal and electron microscopy indicated that Ref(2)P is preferentially found in membrane-free structures in δvps15 mutants, an alternative explanation for the partial Tx solubility of ubiquitin- and Ref(2)P-positive structures may be that smaller accumulations of aggregating proteins are Tx soluble. In any case, a substantial fraction of the ubiquitin- and Ref(2)P positive structures that accumulated in δvps15 mutants were Tx insoluble, strongly suggesting that protein aggregates accumulate in the absence of Vps15. The ultrastructural appearance of these aggregates has striking resemblance to Ref(2)P positive structures found in neuronal tissue of atg8 mutant flies. In both cases, accumulation of vesicular structures surrounding a densely labeled matrix was observed. This might indicate that either the recruitment of autophagic membranes onto or their functional elongation around protein aggregates is dependent on both Atg8 function and PI3K class III activity (Lindmo, 2008).

In conclusion, this study has shown that the PI3K class III co-activator, Vps15, is required for autophagy in Drosophila. δvps15 mutant tissues accumulate Tx-insoluble ubiquitin and Ref(2)P positive structures, indicating a role of Vps15 in autophagic clearance of aggregate-prone proteins. Given that enhanced autophagy can inhibit aggregate-induced neurodegeneration in Huntington models, neuronal-specific stimulation of the Vps34-Vps15 complex might provide a prospective strategy for developing drugs against neurodegenerative diseases (Lindmo, 2008).


Functions of Atg8a orthologs in other species

Phosphatidylethanolamine positively regulates autophagy and longevity

Autophagy is a cellular recycling program that retards ageing by efficiently eliminating damaged and potentially harmful organelles and intracellular protein aggregates. This study shows that the abundance of phosphatidylethanolamine (PE) positively regulates autophagy. Reduction of intracellular PE levels by knocking out either of the two yeast phosphatidylserine decarboxylases (PSD) accelerated chronological ageing-associated production of reactive oxygen species and death. Conversely, the artificial increase of intracellular PE levels, by provision of its precursor ethanolamine or by overexpression of the PE-generating enzyme Psd1, significantly increased autophagic flux, both in yeast and in mammalian cell culture. Importantly administration of ethanolamine was sufficient to extend the lifespan of yeast (Saccharomyces cerevisiae), mammalian cells (U2OS, H4) and flies (Drosophila melanogaster). It is thus postulated that the availability of PE may constitute a bottleneck for functional autophagy and that organismal life or healthspan could be positively influenced by the consumption of ethanolamine-rich food (Rockenfeller, 2015).

Phosphatidylethanolamine (PE) is a phospholipid found in all living organisms. Together with phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylinositol (PI), PE represents the backbone of most biological membranes. PE is the second-most abundant phospholipid in mammalian membranes ranging from 20% to 50%. In yeast, PE is essential for growth and is generated through four different enzymatic pathways: PE can be produced by decarboxylation of PS, as a first option at the mitochondrial membrane via phosphatidylserine decarboxylase 1 (Psd1) or, as a second, option at the Golgi and vacuolar membranes through phosphatidylserine decarboxylase 2 (Psd2). As a third possibility, PE can be produced from actively retrieved extracellular ethanolamine which is cytidine 5'-diphosphate-activated and then coupled to diacylglycerol to generate PE. The fourth, scarcely employed PE-generating pathway is based on the lysophospholipid acylation of lyso-PE. Importantly, PE does not spontaneously assemble in bilayers and rather incorporates into curved structures, such as the inverted hexagonal phase. The physiological function of non-bilayer lipids in membranes is considered to reside in their interaction with membrane proteins via the membrane lateral pressure and membrane tethering and fusion processes, which are relevant for autophagy (Rockenfeller, 2015).

The term 'autophagy' describes a degradation process affecting intracellular components which as an important cytoprotective mechanism, is closely linked to ageing. Autophagy mainly differs from the proteasomal pathway, the other major cellular degradation mechanism, in two aspects. First, autophagy can degrade large particles or whole organelles and second, the final degradation occurs in the lysosome/vacuole and not at the proteasome. Prior to the actual degradation, the cargo is gathered in autophagic particles, which are surrounded by a characteristic double-membrane. However, the origin of these autophagosomal membranes is still controversial and might actually depend on the mode of autophagy induction. Among the discussed membrane sources are the Golgi apparatus, the endosplamic reticulum (ER) or the mitochondrion-associated membrane, which is formed at the interface between the ER. In higher eukaryotes autophagic membranes are enriched in PE with a high degree of unsaturation, similarly to the PE species found in mitochondria. Moreover, the pre-autophagosomal structure or phagophore assembly site (PAS), which appears at the very beginning of autophagosome formation, already harbours Atg9, an autophagy-related transmembrane protein that shuttles between mitochondria and the PAS structure in yeast (Rockenfeller, 2015).

Importantly, PE also functions as an anchor to autophagosomal membranes for the autophagy-related protein Atg8 in yeast and its mammalian orthologue LC3. This PE anchor is provided to LC3/Atg8 post-translationally in a process called lipidation. First, LC3/Atg8 is carboxy-terminally cleaved by proteases from the Atg4 family. Subsequently, the remaining C-terminal glycine is coupled to PE in a series of ubiquitination-like reactions involving diverse Atg-proteins. In vitro, Atg8-PE causes hemifusion of vesicles, which argues for its potential role in autophagosomal phagophore expansion. Consistently, semisynthetic LC3-PE has recently been described to stimulate membrane tethering and fusion. It is thus reasoned that the overall abundance of PE might be critical for PE-lipidation of LC3/Atg8 and could thus regulate autophagosomal membrane formation. Therefore, this study tested whether increasing cellular PE levels might have an impact on autophagy and lifespan regulation (Rockenfeller, 2015).

Knock-out of PSD1 or PSD2 shortens the chronological lifespan of S. cerevisiae, whereas PSD1-overexpression enhances the autophagic capacity and increases longevity. Furthermore, external administration of ethanolamine increases endogenous PE levels, enhances autophagic flux and extends the lifespan of yeast, mammalian cells in culture and flies (Drosophila melanogaster) (Rockenfeller, 2015).

Autophagy is regarded as one of the major cytoprotective mechanisms during ageing, and thus is a crucial process to counteract age-associated pathologies. Age-associated neurodegenerative disorders including Alzheimer's and Parkinson's disease may be postponed or attenuated by chronic induction of autophagy, and there is substantial evidence that genetic or pharmacological induction of autophagy can increase the healthspan and lifespan of multiple model organisms including yeast, worms, flies and mice. These findings have spurred the interest in identifying novel, non-toxic pharmacological inducers of autophagy. So far, several agents have been shown to induce autophagy and increase lifespan across several species, namely rapamycin, resveratrol and spermidine. The present results suggest that ethanolamine might be yet another potent autophagy inducer that promotes longevity. Like rapamycin, resveratrol and spermidine, ethanolamine is a naturally occurring component that is endowed with a favourable toxicity profile (note that the oral LD50 in rats is 1.72g/kg) when compared with rapamycin and resveratrol (Rockenfeller, 2015).

This study provides evidence that ethanolamine-mediated autophagy induction correlates with enhanced longevity in yeast and mammalian cell culture. This is in line with a previous study in yeast demonstrating that PE is a limiting factor for autophagy in a carboxypeptidase Y-defective background. The current results demonstrate that these observations are applicable to a wild-type scenario in yeast too and can be extended to mammalian cell cultures. Still, future experiments will need to clarify if ethanolamine-induced autophagy is beneficial to higher organisms. It was indeed possible to observe a significant increase in the mean lifespan of flies upon supplementation with ethanolamine, but whether this is because of autophagy must be tackled in the follow-up studies (Rockenfeller, 2015).

Besides the pharmacological application of ethanolamine as an anti-ageing agent, the results underline the importance of this molecule for autophagy regulation. In vivo, phosphatidylserine decarboxylase plays a major role in the supply of PE, which is a major component of mitochondrial membranes in yeast and mammals. It is thus not surprising that the abrogation of yeast PSD1 or mammalian PISD affects mitochondrial dynamics such as mitochondrial fusion processes and reduces yeast lifespan. Interestingly, in psd1Δ Rho0 (lacking mitochondrial DNA) the survival rates at day 1 exceed those of the Rho+ counterpart, which suggests that a mitochondrial signal might be involved as a cell death trigger. Similarly, the phenotype of PISD homozygous knock-out mice is so severe that embryos die at day 8 to 10 of embryonic development. In another yeast study Psd2 has been described as being important for PE synthesis and autophagy under conditions of cadmium stress. This is interesting as it generally underlines the importance of PE-abundance for functional autophagy. However, in most of these experiments stronger effects relating to Psd1 function were observed. This study showed that PSD1 overexpression induced autophagy and increased the yeast lifespan. Genetic abrogation of autophagy-related factors in the overexpression setting would in addition strengthen the hypothesis that autophagy was crucial for lifespan extension, which will be tackled in a follow-up study. The yeast and mammalian data indicate that ethanolamine treatment triggers autophagy through a phylogenetically conserved pathway. Moreover, at least in mammalian cells autophagy induction by ethanolamine was neither associated with a decrease in mTORC1 activity, as measured by the phosphorylation of p70s6k, nor with an activation of AMPK. Although it cannot be excluded that alternative regulation processes are involved, it is possible that the availability of PE alone is sufficient to signal autophagy induction (Rockenfeller, 2015).

Of note, lipid determination during chronological ageing, might be diffused to some extent, as lipid extracts are not solely derived from living, but also from dead cells, which might still reside lipolytic activity and thus compromise the results of lipid profiling. However, this should not only affect PE content but similarly other phospholipid classes such as PC, PI or PS. Using the same lipid extracts as for PE-detection, PC-measurement did not reveal significant change for psd2Δ, but slightly decreased PC levels in psd1Δ compared with wild type. Although PC is only reduced by 28% comparing psd1Δ to wild type on day 3 of chronological ageing, the PE reduction measures 77%. Thus, the PE reduction in psd1Δ and psd2Δ is rather interpreted as a predominant consequence of their primary loss of enzymatic function and not as a result of the dead cell population (Rockenfeller, 2015).

One suitable marker of autophagy is the PE-lipidation of LC3/Atg8 as it was used in this study. However, PE-lipidated LC3 has also been shown to localise to lipid droplets and to contribute to their formation. The LC3 conjugation system thus seems to be involved in lipid metabolism. In turn, autophagy has also been shown to be directly involved in lipid turnover. These studies show that lipid metabolism/storage and autophagy share some functional features. It is thus tempting to speculate that PE may act as a crucial molecular (and regulatory) link between autophagy and lipid metabolism (Rockenfeller, 2015).

Altogether, these results have identified ethanolamine as an autophagy-inducing stimulus that will be relevant for future autophagy, lipid and ageing research. Given its anti-ageing potential, ethanolamine might even emerge as a new therapeutic agent in the treatment of age-associated human diseases (Rockenfeller, 2015).

Atg8: an autophagy-related ubiquitin-like protein family

Autophagy-related (Atg) proteins are eukaryotic factors participating in various stages of the autophagic process. Thus far 34 Atgs have been identified in yeast, including the key autophagic protein Atg8. The Atg8 gene family encodes ubiquitin-like proteins that share a similar structure consisting of two amino-terminal alpha helices and a ubiquitin-like core. Atg8 family members are expressed in various tissues, where they participate in multiple cellular processes, such as intracellular membrane trafficking and autophagy. Their role in autophagy has been intensively studied. Atg8 proteins undergo a unique ubiquitin-like conjugation to phosphatidylethanolamine on the autophagic membrane, a process essential for autophagosome formation. Whereas yeast has a single Atg8 gene, many other eukaryotes contain multiple Atg8 orthologs. Atg8 genes of multicellular animals can be divided, by sequence similarities, into three subfamilies: microtubule-associated protein 1 light chain 3 (MAP1LC3 or LC3), gamma-aminobutyric acid receptor-associated protein (GABARAP) and Golgi-associated ATPase enhancer of 16 kDa (GATE-16), which are present in sponges, cnidarians (such as sea anemones, corals and hydras) and bilateral animals. Although genes from all three subfamilies are found in vertebrates, some invertebrate lineages have lost the genes from one or two subfamilies. The amino terminus of Atg8 proteins varies between the subfamilies and has a regulatory role in their various functions (Shpilka, 2011).


REFERENCES

Search PubMed for articles about Drosophila Atg8

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Jain, A., Rusten, T. E., Katheder, N., Elvenes, J., Bruun, J. A., Sjottem, E., Lamark, T. and Johansen, T. (2015). p62/sequestosome-1, Autophagy-related Gene 8, and autophagy in Drosophila are regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), independent of transcription factor TFEB. J Biol Chem 290: 14945-14962. PubMed ID: 25931115

Kim, M., Semple, I., Kim, B., Kiers, A., Nam, S., Park, H. W., Park, H., Ro, S. H., Kim, J. S., Juhasz, G. and Lee, J. H. (2015). Drosophila Gyf/GRB10 interacting GYF protein is an autophagy regulator that controls neuron and muscle homeostasis. Autophagy 11(8): 1358-1372. PubMed ID: 26086452

Kobayashi, M., Itoh, K., Suzuki, T., Osanai, H., Nishikawa, K., Katoh, Y., Takagi, Y. and Yamamoto, M. (2002). Identification of the interactive interface and phylogenic conservation of the Nrf2-Keap1 system. Genes Cells 7(8): 807-820. PubMed ID: 12167159

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Rockenfeller, P., Koska, M., Pietrocola, F., Minois, N., Knittelfelder, O., Sica, V., Franz, J., Carmona-Gutierrez, D., Kroemer, G. and Madeo, F. (2015). Phosphatidylethanolamine positively regulates autophagy and longevity. Cell Death Differ 22(3): 499-508. PubMed ID: 25571976

Rodriguez-Fernandez, I. A., Qi, Y. and Jasper, H. (2019). Loss of a proteostatic checkpoint in intestinal stem cells contributes to age-related epithelial dysfunction. Nat Commun 10(1): 1050. PubMed ID: 30837466

Shpilka, T., Weidberg, H., Pietrokovski, S. and Elazar, Z. (2011). Atg8: an autophagy-related ubiquitin-like protein family. Genome Biol 12(7): 226. PubMed ID: 21867568

Simonsen, A., Cumming, R. C., Brech, A., Isakson, P., Schubert, D. R. and Finley, K. D. (2008). Promoting basal levels of autophagy in the nervous system enhances longevity and oxidant resistance in adult Drosophila. Autophagy 4: 176-184. PubMed ID: 18059160


Biological Overview

date revised: 22 June 2019

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.