Organisms modulate their growth according to nutrient availability. Although individual cells in a multicellular animal may respond directly to nutrient levels, growth of the entire organism needs to be coordinated. This study provides evidence that in Drosophila, coordination of organismal growth originates from the fat body, an insect organ that retains endocrine and storage functions of the vertebrate liver. A genetic screen for growth modifiers discovered slimfast, a gene that encodes an amino acid transporter. Remarkably, downregulation of slimfast specifically within the fat body causes a global growth defect similar to that seen in Drosophila raised under poor nutritional conditions. This involves TSC/TOR signaling in the fat body, and a remote inhibition of organismal growth via local repression of PI3-kinase signaling in peripheral tissues. These results demonstrate that the fat body functions as a nutrient sensor that restricts global growth through a humoral mechanism (Colombani, 2003).
In multicellular organisms, the control of growth depends on the integration of various genetic and environmental cues. Nutrient availability is one of the major environmental signals influencing growth and, as such, has dictated adaptative responses during evolution toward multicellularity. In particular, complex humoral responses ensure that growth and development are properly coordinated with nutritional conditions (Colombani, 2003).
In isolated cells, amino acid withdrawal leads to an immediate suppression of protein synthesis, suggesting that cells are protected by active sensing mechanims that block translation prior to depletion of internal amino acid stores. In many mammalian cell types, changes in amino acid diet affect the binding of the translation repressor 4EBP1 to initiation factor eIF4E and the activity of ribosomal protein S6 kinase (S6K). These two signaling events require the activity of TOR (target of rapamycin), a conserved kinase recently shown to participate in a nutrient-sensitive complex both in mammalian cells and in yeast. Mutations in the Drosophila TOR homolog (dTOR) results in cellular and physiological responses characteristic of amino acid deprivation and establish that TOR is cell autonomously required for growth in a multicellular organism. Furthermore, the TSC (tuberous sclerosis complex) tumor suppressor, consisting of a TSC1 and TSC2 heterodimer (TSC1/2), as well as the small GTPase Rheb participate to the regulation of TOR function. Overall, these data suggest that TSC, Rheb, TOR, and S6K participate in a conserved pathway that coordinates growth with nutrition in a cell-intrinsic manner (Colombani, 2003).
In multicellular organisms, humoral controls are believed to buffer variations in nutrient levels. However, little is known about how growth of individual cells is coordinated. In vertebrates, growth-promoting action of the growth hormone (GH) is mostly relayed to peripheral tissues through the production of IGF-I. Binding of IGF-I to its cognate receptor tyrosine kinase (IGF-IR) induces phosphorylation of insulin receptor substrates (IRS), which in turn activate a cascade of downstream effectors. These include phospho-inositide 3-kinase (PI3K), which generates the second messenger phosphatidylinositol-3,4,5-P3 (PIP3), and thereby activates the AKT/PKB kinase. Genetic manipulation of IGF-I, IGF-IR, PI3K, and AKT in mice modulates tissue growth in vivo thus demonstrating a requirement of the IGF pathway for growth. In Drosophila, both loss- and gain-of function studies have also exemplified the role of a conserved insulin/IGF signaling pathway in the control of growth. Ligands for the unique insulin receptor (Inr) constitute a family of seven peptides related to insulin, the Drosophila insulin-like peptides (Dilps). Remarkably, three dilp genes (dilp2, dilp3, and dilp5) are expressed in a cluster of seven median neurosecretory cells (m-NSCs) in the larval brain, suggesting that they have an endocrine function. Indeed, ablation of the seven dilp-expressing mNSCs in larvae induces a systemic growth defect (Colombani, 2003).
Both in flies and mice, mutations in IRS provoke growth retardation as well as female sterility similar to what is observed in starved animals. Moreover, PI3K activity in Drosophila larvae depends on the availability of proteins in the food. Overall, this supports the notion that the insulin/IGF pathway might coordinate tissue growth with nutritional conditions. However, upon amino acid withdrawal, neither PI3K nor AKT/PKB activities are downregulated in mammalian or insect cells in culture, suggesting that this pathway does not directly respond to nutrient shortage. Hence, an intermediate sensor mechanism must link nutrient availability to insulin/IGF signaling (Colombani, 2003).
An intriguing possibility is that specific organs could function as nutrient sensors and induce a nonautonomous modulation of insulin/IGF growth signaling in response to changes in nutrient levels. This study used a genetic approach in Drosophila to assess both the cellular and humoral responses to amino acid deprivation in the context of a developing organism. The insect fat body (FB) has important storage and humoral functions associated with nutrition, comparable to vertebrate liver and adipose tissue. During larval stages, the FB accumulates large stores of proteins, lipids, and carbohydrates, which are normally degraded by autophagy during metamorphosis in order to supply the developing tissues but can also be remobilized during larval life to compensate transitory nutrient shortage. In addition to its storage function, the FB also has endocrine activity and supports growth of imaginal disc explants and DNA replication of larval brains in coculture. This study demonstrates that the FB operates as a sensor for variations in nutrient levels and coordinates growth of peripheral tissues accordingly via a humoral mechanism (Colombani, 2003).
In the course of a P[UAS]-based overexpression screen for growth modifiers, a P[UAS]-insertion line (UY681) was found to cause growth retardation upon ectopic activation. Sequence analysis revealed that P(UY)681 is inserted in a predicted gene (CG11128) that encodes a putative protein showing strong homology with amino acid permeases of the cationic amino acid transporter (CAT) family. The P[UAS] element is inserted in the first intron of the CG11128 gene, potentially driving transcription of an antisense RNA in a GAL4-dependent manner. To assess the function of this transporter, 3H-arginine uptake was measured in S2 cells. Results indicate that amino acid uptake is either enhanced by transfection of a CG11128 cDNA or suppressed by RNAi, indicating that the encoded protein presents CAT activity. In situ hybridization revealed basal levels of CG11128 expression in most larval tissues but much higher levels in the FB and the gut, two tissues involved in amino acid processing (Colombani, 2003).
By P element remobilization, an imprecise excision was obtained that deletes the sequences encoding the N-terminal half of the protein. 87% of homozygous mutant animals die during larval stages. The few viable adults emerged after a 2 day delay and were smaller and markedly slimmer than control animals. The associated gene was named slimfast (slif) and the excision allele slif1. Weight measurement indicated that homozygous slif1 adult males displayed a 16% mass reduction compared to control. Accordingly, adult wing size was reduced by 8% due to a reduction of both cell size and cell number. When the slif1 allele was in trans to Df(3L)Δ1AK, a deficiency covering the locus, larval lethality was slightly enhanced, suggesting that slif1 corresponds to a strong hypomorphic allele. The amino acid transporter function of slif, as well as the phenotypes observed upon reduction of slif function suggest that slif mutant animals might suffer amino acid deprivation. A major consequence of amino acid deprivation in larvae is the remobilization of nutrient stores in the FB, which typically results in aggregation of storage vesicles. Consistently, fusion of storage vesicles was observed in the FB of slif1 larvae and was indistinguishable from that observed in animals fed on protein-free media (Colombani, 2003).
GAL4 induction of P(UY)681 resulted in a growth-deficient phenotype similar to that of slif1 loss of function. The antisense orientation of P(UY)681 suggested that the growth defect following GAL4 induction was due to an RNAi effect. Indeed, Northern blot analysis revealed that ubiquitous GAL4-dependent activation of P(UY)681 using the daughterless-GAL4 (da-GAL4) driver strongly reduced slif mRNA levels. Only two of the three alternative first exons are potentially affected by the antisense RNA, possibly explaining the residual accumulation of slif mRNAs in da-GAL4; P(UY)681 animals. Most of these animals died at larval stage, similar to what was observed for slif1 mutants. Specific induction of P(UY)681in the wing disc using the MS1096-GAL4 driver provoked a reduction of the adult wing size, which could be either rescued by coactivation of a UAS-slif transgene or enhanced by reducing slif gene dosage with the heterozygous Df(3L)Δ1AK deficiency. Thus, GAL4-dependent activation of P(UY)681 reduces slif function and defines a conditional loss-of-function allele hereafter termed slifAnti (Colombani, 2003).
As expected, loss of slif function using the slifAnti allele also mimicked amino acid deprivation. Accordingly, ubiquitous slifAnti induction in growing larvae resulted in storage vesicle aggregation and strong reduction of global S6 kinase activity, similar to what was reported in animals raised on protein-free diet. Additionally, an increase in PEPCK1 gene transcription was observed, similar to the effect of amino acid withdrawal. In summary, this study has identified two loss-of-function alleles of the slif gene whose defects mimic physiological aspects of amino acid deprivation. Importantly, the conditional slifAnti allele provides a unique tool to mimic an amino acid deprivation in a tissue-specific manner (Colombani, 2003).
This study established that the FB is a sensor tissue for amino acid levels, as downregulation of the Slif amino acid transporter within the FB is sufficient to induce a general reduction in the rate of larval growth. In contrast, specific disruption of slif in imaginal discs, larval gut, or salivary glands did not induce a nonautonomous growth response, suggesting that these tissues do not participate in the systemic control of growth. The dilp-expressing median neurosecretory cells (m-NSCs) also affect growth control, since selective ablation of these cells in the larval brain induces an overall reduction of animal size. In response to complete sugar and protein starvation, the m-NSCs stop expressing dilp3 and dilp5 genes, suggesting that these neurons also sense nutrient levels. This study shows that the selective reduction of slif function in these cells has no obvious effect on tissue growth and animal development. This indicates that the seven dilp-expressing m-NSCs do not constitute a general amino acid sensor. In contrast, the role of m-NSCs in carbohydrate homeostasis and the observation that they stop expressing certain dilp genes when larvae are deprived of sugar rather suggests that these cells have a role in sensing carbohydrate levels (Colombani, 2003 and references therein).
This analysis also provides a framework in which to understand the phenotype of minidisc, a mutation in an amino acid transporter gene that exhibits nonautonomous growth defects in imaginal discs (Colombani, 2003).
In a number of model systems, both PI3K and TOR have been implicated in linking growth to nutritional status and, until recently, were considered as intermediates of a common regulatory pathway. In yeast, the TOR kinase is part of a cell-autonomous nutrient sensor, which controls protein synthesis, ribosome biogenesis, nutrient import, and autophagy. Genetic analysis in Drosophila indicates that dTOR is required for cell-intrinsic growth control. The results obtained using the slifAnti allele in the wing disc indicate that individual tissues have indeed the potential to respond to amino acid deprivation in a cell-autonomous manner. Nonetheless, this study also demonstrates that the TOR nutritional checkpoint participates in a systemic control of larval growth emanating from the FB. Within a developing organism, each cell may integrate these two distinct inputs regarding nutritional status, one originating from a systemically-acting FB sensor, and the other from TOR-dependent signaling in individual cells. One can further speculate that depending on the strength and duration of starvation, different in vivo nutritional checkpoints will be hierarchically recruited to protect the animal and that the systemic control might, in most physiological situations, override the cell-autonomous control. Indeed, as the data demonstrate, the FB sensor is sufficient to induce a general and coordinated response to starvation without calling individual cell-autonomous mechanisms into play (Colombani, 2003).
Several lines of evidence indicate that the PI3K pathway is not part of the sensor mechanism in FB cells. First, a sensor for PI3K activity in the FB is only marginally affected by amino acid deprivation in that tissue, indicating that the cell-autonomous response to amino acid starvation does not directly influence PI3K signaling. This is reminiscent of previous observations in mammalian cultured cells, showing that PI3K activity does not respond to variations in amino acid levels. Moreover, inhibition of PI3K signaling by dPTEN expression in the FB is not sufficient to trigger the sensing mechanism. Although, dPTEN overexpression causes a complete disappearance of the PI3K sensor accompanied by growth suppression of FB cells, the FB maintains a critical mass that allows for normal larval growth. In contrast, the regulatory subunit p60 whose overexpression potently inhibits PI3-kinase in flies has been shown to induce a systemic effect on larval growth when overexpressed in the FB using an Adh-Gal4 driver. This study found that a pumpless ppl-GAL4-directed expression of p60 also provokes a strong suppression of larval growth and a dramatic inhibition of FB development in young larvae. Thus, the systemic effect on growth observed upon p60 overexpression most likely results from a drastic reduction of FB mass, which then fails to support normal larval growth (Colombani, 2003).
These results further indicate that PI3K signaling is a remote target of the humoral message that originates from the FB in response to amino acid deprivation. This is in agreement with previous data showing that PI3K activity is downregulated by dietary amino acid deprivation and explains why global PI3-kinase inhibition mimics cellular and organismal effects of starvation. The existence of a humoral relay reconciles these in vivo studies with the absence of direct PI3K responsiveness to amino acid levels (Colombani, 2003).
The relative resistance of imaginal disc growth to the systemic control exerted by the FB correlates with maintenance of PI3K activity in these tissues. This is in agreement with previous observations that cells in the larval brain and in imaginal discs maintain a slow rate of proliferation under protein starvation, while larval endoreduplicating tissues (ERTs) arrest. This difference might be attributed to the basal levels of dilp2 expression observed in imaginal discs, allowing a moderate growth rate of these tissues through an autocrine/paracrine mechanism. It was recently shown that clonal induction of PI3K potently induces cell-autonomous growth response even in fasting larvae, indicating that some nutrients are still accessible to support cell growth within a fasted larva. The main function of a general sensor could be to preserve these limited nutrients for use by high priority tissues. In this context, local PI3K activation through an autocrine loop in imaginal tissues could favor the growth of prospective adult structures in adverse food conditions. Thus, the FB would have an active role in controlling the allocation of resources depending on nutritional status. In this respect, it is noteworthy that FB cells are relatively resistant to the FB-derived humoral signal, since the PI3K sensor is not drastically affected in the FB of ppl>slifAnti animals. Thereby, essential regulatory functions of the FB could be preserved even in severely restricted nutritional conditions (Colombani, 2003).
How does the FB signal to other tissues? This study suggests that a humoral signal relays information from the FB amino acid sensor and systemically inhibits PI3K signaling. In addition, this downregulation is not due to a direct inhibition of dilp expression by neurosecretory cells in the brain. Nevertheless, it cannot be ruled out that the secretion of these molecules is subjected to regulation in the mNSCs. Both in vivo and in insect cell culture, several imaginal discs growth factors (IDGF) secreted by the FB have been proposed to function synergistically with Dilp signaling to promote growth. However, this study did not find any modification of IDGF expression in the FB of larvae raised on water- or sugar-only diet, or upon FB induction of slifanti. In vertebrates, the different functions of the circulating IGF-I are modulated through its association with IGF-BPs and acid labile subunit (ALS). In particular, the formation of a ternary complex with ALS leads to a considerable extension of IGF-I half-life. The finding that a Drosophila ALS ortholog is expressed within the FB in an amino acid-dependent manner provides a new avenue to study the molecular mechanisms of nonautonomous growth control mediated by the FB (Colombani, 2003).
This study highlights the contribution that genetics can provide to unravel the mechanisms of physiological control. Using a genetic tool to mimic amino acid deprivation, it was demonstrated that nutrition systemically controls body size through an amino acid sensor operating in the FB. It is proposed that (1) in metazoans, a systemic nutritional sensor modulates the conserved TOR-signaling pathway, and (2) the response to sensor activation is relayed by a hormonal mechanism, which triggers an Inr/PI3K-dependent response in peripheral tissues (Colombani, 2003).
In many species, reducing nutrient intake without causing malnutrition extends lifespan. Like DR (dietary restriction), modulation of genes in the insulin-signaling pathway, known to alter nutrient sensing, has been shown to extend lifespan in various species. In Drosophila, the target of rapamycin (TOR) and the insulin pathways have emerged as major regulators of growth and size. Hence, the role of TOR pathway genes in regulating lifespan has been examined by using Drosophila. Inhibition of TOR signaling pathway by alteration of the expression of genes in this nutrient-sensing pathway, which is conserved from yeast to human, extends lifespan in a manner that may overlap with known effects of dietary restriction on longevity. In Drosophila, TSC1 and TSC2/Gigas (tuberous sclerosis complex genes 1 and 2) act together to inhibit TOR (target of rapamycin), which mediates a signaling pathway that couples amino acid availability to S6 kinase, translation initiation, and growth. Overexpression of dTsc1, dTsc2, or dominant-negative forms of dTOR or dS6K all cause lifespan extension. Modulation of expression in the fat is sufficient for the lifespan-extension effects. The lifespan extensions are dependent on nutritional condition, suggesting a possible link between the TOR pathway and dietary restriction (Kapahi, 2004).
The Drosophila homologs of human Tsc1 (Hamartin) and Tsc2 (tuberin) function in vivo as a complex that controls growth and size in a cell-autonomous manner. To examine their role in regulating lifespan, dTsc1 and dTsc2 were overexpressed through the ubiquitously expressed driver, daughterless (da-GAL4). Overexpression in transgenic flies carrying UAS constructs containing dTsc1 or dTsc2 extends mean lifespan at 29°C by 14% and 12%, respectively. Since GAL4 enhancer traps generally yield stronger effects at 29°C, most of the experiments were performed at that temperature (Kapahi, 2004).
dTsc1 and dTsc2 physically interact with dTOR, which is conserved from yeast to human as a nutrient sensor. Loss of dTsc1 in Drosophila eye leads to an increase in cell size, provided that dTOR is present. Surprisingly, however, dTOR overexpression causes a reduction in cell size, a phenotype similar to dTOR loss-of-function mutations, perhaps due to titration of cofactors required for TOR signaling. The effect of dTOR on lifespan was examined by using three UAS. One carries the full-length wild-type TOR gene. The second carries FRB, the 11 kDa FKBP12-rapamycin binding domain, which has been shown to prevent S phase entry when injected into human osteosarcoma cells. The third carries TED (toxic effector domain), containing the 754 amino acid central region, which inhibits cell growth and arrests cells in G1 when overexpressed in yeast (Hennig, 2002). Ubiquitous overexpression with the da-GAL4 driver of UAS-dTORFRB led to a mean lifespan increase at 29°C of 24%. However, overexpression of UAS -dTORWT or UAS-dTORTED prevented eclosion to adulthood (Kapahi, 2004).
S6 kinase activation upon phosphorylation has been implicated in mediating the downstream effects of TOR on translation initiation in flies and mammals. S6 kinase phosphorylation of ribosomal protein S6 is accompanied by upregulation of a class of mRNAs containing an oligopyrimidine tract at their transcriptional start site termed 5'TOP (Thomas, 2002). Some 200 genes, most of which encode components of the translational apparatus including ribosomal proteins and elongation factors, have this sequence and can account for about 20% of total cellular mRNA. Flies carrying homozygous mutations in dS6K show a developmental delay and a reduction in body size. The stimulation of dS6K phosphorylation by dTOR is abrogated when dTsc1 and dTsc2 are overexpressed. Furthermore, flies with reduced dTSC1 show increased dS6 kinase activation, and genetic reduction of S6 kinase level can rescue the lethality caused by loss of function of dTsc1 (Kapahi, 2004).
The role of S6 kinase in regulating lifespan was examined by using dominant-negative and constitutively active constructs. The dominant-negative effect was achieved by replacing the conserved lysine in the ATP binding site by glutamine (UAS-dS6KKQ), which causes cell-size reduction. The constitutively active form was generated by replacing the phosphorylation sites of S6 kinase by acidic amino acids (UAS-dS6KSTDETE), causing an autonomous cell size increase. By using da-GAL4 to drive ubiquitous overexpression of the dominant-negative form, a mean lifespan increase of 22% at 29°C was observed. Conversely, overexpression of the constutively active form of S6 kinase caused a mean lifespan decrease of 34% at 29°C. Overexpression of dTsc2 and dTORFRB was also tested at 25°C and led to a 20% and 26% increase in mean lifespan increase, respectively (Kapahi, 2004).
To determine which tissues are responsible for the lifespan extension, various GAL4 drivers with specific GAL4 expression pattern were employed to overexpress dTsc2 via a UAS promoter. Overexpression in the eye by using the driver gmr-GAL4 or in the nervous system by using appl-GAL4 did not extend lifespan. In contrast, by using the drivers 24B-GAL4 and PO188-GAL4, enhancer traps that are predominantly expressed in the muscle and fat, results in mean lifespan extensions of 27% and 37%, respectively, at 29°C. The fat-specific drivers DJ634-GAL4 and PO163-GAL4, when used to overexpress dTsc2, also led to a mean lifespan extension of 22% and 31%, respectively, at 29°C. Using DJ634-GAL4 to overexpress the dominant-negative form of TOR (UAS-dTORFRB) or of S6 kinase (UAS-UAS-dS6KKQ) also led to mean lifespan increases of 30% and 29%, respectively, at 29°C. These results indicate that manipulation of the TSC, TOR, and S6 kinase genes in the fat tissue is sufficient for their lifespan extension effects in Drosophila (Kapahi, 2004).
Amino acids have been shown to activate dS6k via TOR, an effect that can be abrogated in the presence of increased levels of dTsc1 and dTsc2. Since nutrients in the diet can modulate lifespan and because the TOR pathway is a critical mediator of nutrient signaling, it was asked whether the observed lifespan-extension effects are dependent on nutrient conditions. This was tested with overexpression of dTsc2 by using the ubiquitously expressing da-GAL4 driver. Flies were allowed to develop to adulthood under standard laboratory food and then maintained on specially prepared food containing various concentrations of yeast extract. At high concentrations of yeast extract, which may be regarded as the opposite of dietary restriction, the lifespan of control flies (da-GAL4/+) is severely reduced. However, overexpression of dTsc2 protects the fly from the deleterious effects of rich food, as if mimicking the effect of dietary restriction. Similar results were observed by overexpression of the dominant-negative form of S6 kinase (Kapahi, 2004).
Recent evidence from Drosophila suggests that signaling through TSC is both parallel to and interacting with the insulin pathway. This is supported by the finding that heterozygosity of dTsc1 or dTsc2 is sufficient to rescue the lethality of loss-of-function dInR mutants. However, the finding that loss-of-function mutations of dTsc1 and dPTEN, a phosphatase that negatively regulates the insulin-signaling pathway, cause cell autonomous and additive increases in cell size suggests that they may be in parallel pathways. Furthermore, in Drosophila, dPTEN loss of function, which leads to an increase in cell size, is only slightly suppressible by loss of function of dFOXO, a fly homolog of C. elegans daf-16. However, the increase in cell size resulting from dTsc1 is enhanced by dFOXO loss of function. Interestingly, unlike long-lived daf-2 mutants, the lifespan extension due to TOR deficiency in C. elegans is not suppressible by a daf-16 mutation. However, the TOR mutant animals do not further extend lifespan in a daf-2 background, leading to the possibility that TOR may be acting downstream or separately from daf-16 to exert its lifespan effects (Kapahi, 2004).
Lifespan extension has been linked with other phenotypes, including stress resistance, metabolic rate, lipid level, reproductive capacity, and body size. The long-lived strains described above with their respective controls for resistance to starvation were compared but no significant differences were found. Similarly, no significant differences were observed for weight and lipid content among these strains. It may be that lifespan extension can be produced by mild modulation of these genes, whereas effects on other phenotypes require severe perturbations. While lifespan extension is observed by using the da-GAL4 driver to overexpress dTsc1 or dTsc2 alone, simultaneous overexpression of dTsc1 and dTsc2 prevented eclosion to adulthood. Similarly, no change in size is observed if dTsc1 or dTsc2 alone are overexpressed in the eye, but a cell-autonomous decrease in size is seen when both are overexpressed simultaneously. Lifespan extension by chico is semidominant, but its effect on body size is recessive. Dominant effects on lifespan are observed with the genes Inr, EcR, Indy, and Rpd3, but their effects on lifespan can be uncoupled from other phenotypes such as fecundity, stress resistance, or lipid accumulation (Kapahi, 2004).
In humans, mutations in TSC1 and TSC2 lead to tuberous sclerosis, a common disorder characterized by the presence of benign tumors in various tissues, with some having large cells. DR in mice has been shown to protect against age-related tumorigenesis. These results suggest a link between lifespan extension by DR and the activities of genes in the TOR pathway. Hence, it is conceivable that the protective effects of DR on tumorigenesis and age-related decline might come from inhibition of such nutrient-responsive pathways (Kapahi, 2004).
These results show that upregulation of dTsc2 in the fat is sufficient for lifespan extension effects in Drosophila. Reduction of daf-2 levels in the C. elegans nervous system has been shown to be sufficient for lifespan extension. However, the lifespan extensions due to mutations in the insulin pathway or germline ablation in C. elegans are dependent on daf-16 activity in the intestine, the fat storage tissue in C. elegans. In Drosophila, the fat body has been proposed to modulate insulin signaling in peripheral tissues by secretion of dALS (acid-labile subunit), which, in mammals, forms a ternary complex with insulin-like growth factor, leading to an extension of the half-life of its ligand. Recently, mice with FIRKO (fat-specific insulin receptor knockout) have been shown to live 18% longer than controls . Hence, it is possible that secondary endocrine signals downstream of the insulin and TOR signaling pathways are released from the fat, and these affect the rate of aging in other tissues. Juvenile hormone and ecdysone are two such endocrine signals that have been implicated in regulating lifespan in conjunction with the insulin pathway in Drosophila (Kapahi, 2004).
The insulin/PI3K signaling pathway controls both tissue growth and metabolism. Melted has been identified as a new modulator of this pathway in Drosophila. Melted interacts with both Tsc1 and Foxo and can recruit these proteins to the cell membrane. Evidence is provided that in the melted mutant, Tor activity is reduced and Foxo is activated. The melted mutant condition mimics the effects of nutrient deprivation in a normal animal, producing an animal with 40% less fat than normal (Teleman, 2005).
As a means to identify possible functions of Melted, the Eukaryotic Linear Motif server) was used to look for functional motifs conserved between fly and human Melted. The only conserved motifs found in the N-terminal region of these proteins were two Forkhead-associated domain ligand domains (LIG_FHA_1). Forkhead transcription factors FoxA2, FoxA3, FoxC2, and FoxO1 are involved in glucose and fat metabolism. Insulin signaling activates Akt, which phosphorylates Foxo and leads to its retention in the cytoplasm. It was therefore asked if Melted affects the subcellular localization of a Foxo-GFP fusion protein. Foxo-GFP is predominantly nuclear in the absence of insulin stimulation in serum-starved S2 cells and increases in the cytoplasm after insulin stimulation. In serum-starved cells cotransfected to express Melted, Foxo-GFP is still primarily nuclear, but much of the nonnuclear protein appears at the membrane colocalized with Melted. Upon insulin stimulation, a robust increase in the level of Foxo-GFP was observed at the cell membrane. The interaction was confirmed by coimmunoprecipitation of Melted with Foxo in insulin-stimulated S2 cells (Teleman, 2005).
The observation that insulin stimulation induces a shift toward membrane localization of Foxo in the presence of Melted in S2 cells raised the possibility that melted regulates Foxo activity in vivo. To address this, expression of the Foxo target 4E-BP was examined in wild-type and melted mutant animals. Under fed conditions, insulin signaling is active and 4E-BP transcript levels are relatively low. In wild-type flies that were starved for 24 hr to reduce insulin levels and thereby activate Foxo, 4E-BP transcript increased ~4-fold. In starved flies lacking Melted, 4E-BP transcript increased over 25-fold. This increase in 4EBP transcription was absent in the starved melted/Foxo double mutant, confirming that it is Foxo dependent. Thus, in the absence of Melted, Foxo activity is higher than normal, suggesting that Melted limits Foxo activity in vivo (Teleman, 2005).
To determine whether the elevated Foxo activity observed in melted mutants contributes to the lean phenotype of these animals, the normalized triglyceride levels of melted mutant and melted foxo double-mutant flies were compared. Reducing Foxo activity suppresses the leanness of the melted mutant to a considerable degree, reaching near normal fat levels. The rescue was highly statistically significant. foxo mutants did not show higher-than-normal fat levels compared to wild-type. These observations suggest that Melted acts by regulating Foxo activity to control expression of genes important in fat metabolism (Teleman, 2005).
The Tor pathway integrates information on cellular nutritional status and stress from the heterodimeric Tsc1/2 complex. melted mutants exhibit reduced Tor activity. By recruiting Foxo to the membrane in an insulin-regulated manner Melted influences expression of Foxo targets. By reducing Tor activity and at the same time increasing Foxo activity, the melted mutant mimics the effects of nutrient deprivation in a normal animal, producing a lean phenotype (Teleman, 2005).
To determine whether Tor activity affects fat accumulation, the effects were tested of increasing Tor activity in wild-type and melted mutant adipose tissue. Use was made of a UAS-Tor transgene that can provide Tor activity in vivo when expressed at appropriate levels. It was confirmed that expression of UAS-Tor under ppl-Gal4 control in adipose tissue leads to increased total body fat, as does increasing PI3K activity. In contrast, a comparable elevation of Tor expression in melted mutant flies has no effect on fat levels. Both this result and the significant rescue caused by removal of Foxo indicate that in the melted mutant, the Foxo branch of the pathway becomes limiting for fat accumulation. In view of this finding, it was next asked whether elevated Tor pathway activity could increase fat levels in the melted mutant if Foxo activity was simultaneously reduced. To do so, use was made of the catalytic subunit of PI3K (Dp110) to inactivate Foxo and simultaneously activate Tor. The fat body driver lsp2-Gal4 or the UAS-Dp110 transgenes have little effect on their own in the melted mutant background, but when combined, the elevated PI3K activity in the fat body increases fat levels of the melted mutant. The effect is stronger than that of removing Foxo only, increasing fat levels to above normal. Taken together, these observations suggest that the Tor branch of the pathway contributes to the control of fat levels under conditions in which Foxo activity levels are low. This is normally the case in feeding animals in which insulin levels are relatively high (Foxo activity is elevated under starvation conditions: as seen by comparing 4E-BP levels in fed versus starved wild-type and foxo mutant flies). Under conditions in which insulin levels are low or in the melted mutant, in which Foxo activity is elevated, the effects of Foxo appear to dominate (Teleman, 2005).
The TOR (target of rapamycin) ser/thr protein kinase is the central component of a eukaryotic signaling pathway that regulates growth and is the direct target of the clinically useful drug rapamycin. Recent efforts have identified at least two multiprotein complexes that contain TOR, but little is known in higher eukaryotes about the genes downstream of TOR that control growth. By combining the use of a small molecule inhibitor (rapamycin), transcriptional profiling, and RNA interference in Drosophila tissue culture cells, genes have been identified whose expression responds to Drosophila TOR (dTOR) inhibition and that regulate cell size. Several of the dTOR-regulated genes that function in cell size control have additional roles in cell division. Most of these genes are conserved in mammals and several are linked to human disease. This set of genes is highly enriched for regulators of ribosome biogenesis, which emphasizes the importance of TOR-dependent transcription in building the protein synthesis machinery in higher eukaryotes. In addition, two dTOR-regulated genes, CG3071 and CG6677, have been identified whose human orthologs, SAW and ASH2L, are also under TOR-dependent transcriptional control and encode proteins with conserved functional roles in growth. It is concluded that combining RNA interference with genomic analysis approaches, such as transcriptional profiling, is an effective way to identify genes functioning in a particular biological process. Moreover, this strategy, if applied in model systems with simpler genomes, can identify genes with conserved functions in mammals (Guertin, 2006).
This study defines growth as an increase in cell mass, which is distinct from cell division, although the two processes are coordinated. How coordination is achieved is still debated, but in many cells, cell-cycle progression requires growth. From this model has emerged a hypothesis that a 'size threshold' exists that prevents cell-cycle progression until a critical size is attained. When assessing size, it is not clear what cells measure, but the rate of protein synthesis is a good prediction. Therefore, it is not surprising that rapamycin treatment (which is thought to mimic nutrient deprivation) would change the expression of genes with roles in various aspects of setting the protein synthesis rate (Guertin, 2006).
Studies in Saccharomyces cerevisiae investigating the role of TOR in transcription have determined that yeast TOR regulates expression of metabolic pathway genes, and in addition, ribosomal RNA and ribosomal protein genes; transcription, translation, and replication factors; and protein degradation genes. Studies with mammalian cells have indicated that rapamycin affects expression of a diverse array of genes functioning in nutrient and protein metabolism. But in these studies, a functional connection to growth was not established. Ribosomal proteins and ribosome assembly factors have a critical role in controlling growth. Furthermore, two proteins (Sfp1 and Sch9—an AGC kinase related to AKT and S6K) that activate expression of transcriptional units encoding ribosomal proteins and ribosome assembly factors are regulated in part by TOR. This study study suggests that in higher eukaryotes, TOR regulates expression of genes functioning in multiple steps in the ribosome assembly pathway. But importantly, it was found that many rapamycin-responsive genes that are necessary for normal growth encode ribosome biogenesis regulators, a connection not made in higher organisms (Guertin, 2006).
Previous genetic studies in Drosophila were consulted to determine if any of the genes identified in this study by a cell culture-based system have previously been shown to have a role in organismal growth. One positive growth gene that was identified was CG3333/Nop60B. Partial loss of Nop60B (also named minifly) function results in severe reduction in body size and developmental delay (Giordano, 1999). One positive growth and proliferation gene identified, CG6375/pitchoune/pit, is also required for cell growth and proliferation in developing Drosophila larvae (Zaffran, 1998). Finally, CG5786/ppan (peter pan), a gene identified in a screen for larval growth regulators, was also found to be required for normal growth and proliferation of cultured cells (Migeon, 1999). These findings suggest that this approach led to the identification of physiologically relevant genes (Guertin, 2006).
Nutrient control of gene expression and growth has emerged as a principal concern in the modern era as diet-induced diseases become more prevalent. It is believed that mTOR is at the core of an ancient growth pathway that senses nutrient levels, particularly amino acids, and that rapamycin treatment mimics a 'starvation-like' state. A previous study employing Affymetrix microarrays identified a set of genes in developing Drosophila larvae that respond to starvation. 42% of the 19 genes identified by Zinke (2002) as responding negatively to amino acid starvation were also rapamycin-sensitive genes: a remarkable similarity considering the difference in source material and statistical analysis. If all the genes whose expression decreased significantly (p < 0.01) after rapamycin treatment are considered, it was found that 68% of the genes in the list of Zinke (2002) are also in the current list. While none of the genes Zinke identified as significantly increasing expression upon starvation (14 total) passed cutoffs to be functionally analyzed in this study, 4 of them (28%) did significantly increase expression in response to rapamycin. One of those genes encodes Thor, the Drosophila 4E-BP ortholog. Both the Zinke study and the current study used early versions of the Drosophila Affymetrix chip ('DrosGenome1'), and therefore there might be additional nutrient- and rapamycin-sensitive genes to be discovered (Guertin, 2006).
An appreciation for the role of mTOR in tumorigenesis has emerged from clinical trials indicating that rapamycin might be an effective treatment for some cancers. A general idea is that upregulated mTOR signaling provides tumors with a growth advantage by promoting translation initiation through S6K1 and 4E-BP1. However, it cannot be ruled out that other mTOR-regulated process such as transcription and autophagy are relevant in cancer and other diseases. These processes are conserved in yeast and represent ancient functions of TOR. Perhaps mTOR-regulated genes could be important targets for antigrowth drugs. The fact that many of the growth genes identified block the TSC2 RNAi-induced cell size increase when cosilenced supports such a notion (Guertin, 2006).
Genes with specific links to human disease were also identified in this report. Nop60B (CG3333) is the Drosophila ortholog of human DKC1 (dyskerin), the gene mutated in dyskeratosis congenita (DC). DC is a rare X-linked recessive disease initially characterized by nail dystrophy, abnormal skin pigmentation, mucosal leucoplakia, and premature aging, with patients often succumbing to bone marrow failure before the age of 30. DKC1 encodes a pseudouridine synthase, which associates with box H/ACA small nuclear RNAs and posttranscriptionally modifies rRNA by converting uridine to pseudouridine. In yeast, the DKC1 ortholog (Cbf5p) associates with Nhp2p, the Drosophila ortholog of which (CG5258) was also identified in the screen. Patients with DC are predisposed to tumor formation, and this is mimicked in a mouse model in which half of Dkc1 mutant animals develop tumors. It is perhaps paradoxical that mutations apparently compromising ribosome function promote tumorigenesis. However, a screen for cancer genes in zebrafish identified several ribosomal protein genes as haploinsufficient tumor suppressors, suggesting that ribosome dysfunction may have an important but undefined role in promoting tumor formation (Guertin, 2006).
The human ash2/CG667 gene product (ASH2L) was discovered in a histone methyltransferase (HMT) complex with the tumor suppressor menin (see Drosophila Menin-1). Menin is encoded by the MEN1 gene, which is mutated in familial multiple endocrine neoplasia type 1. Several MEN1 point mutations found in tumors are associated with reduced HMTase activity of the complex. Another report found ASH2L associated with a HMT complex containing the Leukemia protooncoprotein MLL, the human ortholog of Drosophila trithorax, in addition to menin. Interestingly, Drosophila ash2 mutant cells in genetic mosaics exhibit defective cell differentiation and increased cell size, consistent with these conclusions. In another report, Drosophila ASH2 localized to the nucleolus, suggesting that ASH2 might have a role in rDNA transcription. The yeast ortholog of ASH2 is part of the SET1 complex, which is reported to repress rDNA transcription by promoting H3 Lys4 methylation of rDNA. Elucidating the function of ASH2 in cell growth and differentiation might uncover clues to understanding the tumor-suppressor functions of these HMT complexes (Guertin, 2006).
This study combined gene expression profiling with functional analysis by RNAi to identify Drosophila genes that are responsive to acute rapamycin treatment and that regulate cell growth and proliferation. This approach allowed functional annotation of 54 Drosophila genes. Most of the genes have orthologs in species ranging from yeast to mammals, and some are implicated in human disease. With genome-scale RNAi libraries becoming available in many organisms, it is concludes that similar combinatorial approaches might be useful in determining subsets of 'enriched' genes that could be functionally analyzed by targeted RNAi. This study found a rapamycin-sensitive growth-gene set in Drosophila cultured cells. In addition to emphasizing the role of TOR-dependent transcription in growth, human genes were identified with similar transcriptional and functional roles. It is further concluded that similar approaches in model systems with comparatively simpler genomes can be an effective way to predict human gene function (Guertin, 2006).
Target of rapamycin (TOR) is a central regulator of cellular and organismal growth in response to nutrient conditions. In a genetic screen for novel TOR interactors in Drosophila, the clathrin-uncoating ATPase Hsc70-4, which is a key regulator of endocytosis, was identified. Genetic evidence is presented that TOR signaling stimulates bulk endocytic uptake and inhibits the targeted endocytic degradation of the amino acid importer Slimfast. Thus, TOR simultaneously down-regulates aspects of endocytosis that inhibit growth and up-regulates potential growth-promoting functions of endocytosis. In addition, disruption of endocytosis leads to changes in TOR and phosphatidylinositol-3 kinase activity, affecting cell growth, autophagy, and rapamycin sensitivity. These data indicate that endocytosis acts both as an effector function downstream of TOR and as a physiologically relevant regulator of TOR signaling (Hennig, 2006; full text of article).
Inactivation of TOR causes an inhibition of cellular growth, a reduction in cell size, and a suppression of cell cycle progression. In addition to well described changes in protein synthesis and ribosome biogenesis, recent studies have suggested that other cell processes are likely to contribute to these growth effects of TOR. The present study identifies endocytosis as one such process. These results demonstrate that the clathrin-uncoating ATPase Hsc70-4 interacts genetically with TOR and Tsc1, and that bulk endocytosis is stimulated in cells with activated TOR signaling. Conversely, it was found that TOR activity inhibits the endocytic degradation of nutrient transporters such as Slimfast. Together, these endocytic effects of TOR promote both the bulk and targeted uptake of nutrients and other biomolecules required for cell mass increase. In addition to this direct role in cellular biosynthesis and growth, nutrients also act as potent regulators of TOR signaling. Indeed, Slimfast has been identified as an upstream activator of TOR (Colombani, 2003). These findings that disruption of endocytosis effects cell size, rapamycin sensitivity, and TOR kinase activity are consistent with an additional role for endocytosis upstream of TOR (Hennig, 2006).
Mutations that disrupt endocytosis are likely to have both positive and negative effects on nutrient uptake and cell growth because they inhibit bulk endocytic uptake, as well as degradation of nutrient transporters and other signaling molecules. Thus, the overall effects of endocytic disruption on nutrient uptake, cell growth, and TOR signaling are difficult to predict a priori. The results suggest that both the cellular context and the specific step at which endocytosis is blocked influence the growth response. Thus, in fat body cells, expression of ShiK44A resulted in an increase in cell size, whereas loss of Hsc70-4 function caused reduced cell size. It is noted that these changes mirror the effects of these mutations on Slimfast levels; whereas both ShiK44A expression and Hsc70-4 mutation decreased bulk endocytic uptake, only ShiK44A resulted in increased levels of Slimfast. In contrast, both ShiK44A and Hsc70-4 mutants led to the increased size of wing imaginal disc cells, suggesting that in these cells the growth-inhibitory effects of endocytic degradation of membrane proteins such as Slimfast predominate over the potential positive effects of increased bulk uptake. Similarly, the results indicate a complex effect of endocytosis on TOR signaling. Partial reduction in Hsc70-4 levels lead to an increase in TOR signaling, as was evident in an eyTOR interaction and rapamycin resistance. In contrast, larvae that are homozygous mutant for Hsc70-4 show a decrease in TOR kinase activity. These results suggest that modest inhibition of endocytosis may increase TOR signaling, whereas a complete block of endocytosis may reduce it (Hennig, 2006).
A striking parallel to the inverse regulation of bulk and targeted endocytic processes by TOR can be observed in its effects on autophagy in yeast. Through autophagy, random portions of cytoplasm are nonselectively engulfed within double membrane–bound vesicles for delivery to the lysosome. Activation of TOR causes this nonselective form of autophagy to be suppressed, and, instead, the autophagic machinery engages in a selective type of autophagy known as the cytoplasm–vacuole targeting (CVT) pathway, which is responsible for lysosomal delivery of specific hydrolases. Thus, TOR acts as a switch between selective and nonselective autophagy. TOR may also be involved in switching between clathrin-and caveolae/raft-mediated endocytosis in higher eukaryotes. A genome-wide survey of protein kinases found that RNAi-mediated inactivation of TOR in HeLa cells inhibited clathrin-dependent processes such as transferrin uptake and vesicular stomatitis virus infection, and stimulated cavelolae/raft-dependent events (Pelkmans, 2005). Together, these findings suggest that TOR may control the specificity of membrane trafficking components. In addition, the results show that S6K, which is an important TOR substrate, acts downstream of TOR in promoting bulk endocytosis, but is not involved in the suppression of starvation-induced autophagy (Hennig, 2006).
The identification of endocytosis as a TOR-controlled function adds to the growing list of cell processes regulated by TOR, including protein synthesis, ribosome biogenesis, autophagy, metabolic gene expression, and cytoskeletal organization. How these distinct functions interact to achieve a coordinated growth response is only beginning to be understood. One likely mechanism involves the common use of molecular components and cellular substrates by different cell functions, as in the case of selective and nonselective autophagy, bulk endocytosis, and endocytic degradation. Two or more distinct branches of TOR signaling may also act cooperatively to control the same target, as in the case of Slimfast regulation by both translation and endocytosis, or may act in opposition, as previously observed for the role of S6K in limiting autophagy. Finally, distinct TOR complexes may converge on the same targets with opposing effects, as in the regulation of Akt by TOR-raptor versus TOR–rictor complexes (Shah, 2004; Sarbassov, 2005). The finding that TOR signaling regulates the levels of Slimfast, which was previously shown to function upstream of TOR, adds another layer of complexity to the TOR signaling network (Hennig, 2006).
Previous studies have demonstrated reexpression of cell-cycle markers within postmitotic neurons in neurodegenerative tauopathies, including Alzheimer's disease (AD). However, the critical questions of whether cell-cycle activation is causal or epiphenomenal to tau-induced neurodegeneration and which signaling pathways mediate cell-cycle activation in tauopathy remain unresolved. Cell-cycle activation accompanies wild-type and mutant tau-induced neurodegeneration in Drosophila, and genetically interfering with cell-cycle progression substantially reduces neurodegeneration. The data support a role for cell-cycle activation downstream of tau phosphorylation, directly preceding apoptosis. Accordingly it is shown that ectopic cell-cycle activation leads to apoptosis of postmitotic neurons in vivo. As in AD, TOR (target of rapamycin kinase) activity is increased in this model and is required for neurodegeneration. TOR activation enhances tau-induced neurodegeneration in a cell cycle-dependent manner and, when ectopically activated, drives cell-cycle activation and apoptosis in postmitotic neurons. TOR-mediated cell-cycle activation causes neurodegeneration in a Drosophila tauopathy model, identifying TOR and the cell cycle as potential therapeutic targets in tauopathies and AD (Khurana, 2006).
It was first determined whether cell-cycle activation accompanied neurodegeneration in a fly model of tauopathy. Expression of a mutant form of tau linked to familial frontotemporal dementia, tauR406W, in the fly brain (panneural driver: ELAV-GAL4) leads to progressive neurodegeneration (Wittmann, 2001). At eclosion, the brains of tau-expressing flies appear morphologically normal, but by 10 days clear neurodegeneration is observed, characterized histologically by condensation and fragmentation of neuronal nuclei and vacuolization. TUNEL staining identified apoptotic neurons in tau transgenic animals but not in age-matched controls (Khurana, 2006).
PCNA and PH3 were immunostained to assess early and late cell-cycle activation. Control animals were completely negative for PCNA and the M-phase marker phosphohistone-3 (PH3) at 10 days and 30 days. In contrast, brains from tau transgenic flies showed prominent expression of both PCNA and PH3 at 10 days. PCNA staining was particularly prominent in areas of neurodegeneration, as indicated by characteristic nuclear changes and cytoplasmic vacuolization. Together, these findings demonstrate that abnormal activation of the cell cycle accompanies tau-induced apoptotic neurodegeneration in Drosophila and suggest that cell-cycle activation is likely to be a relatively late event in this model (Khurana, 2006).
This study established a causal relationship between cell-cycle activation and tau-induced neurodegeneration in vivo. Expression of both mutant and wild-type tau induce cell-cycle activation in this model, and genetic inhibition of the cell cycle substantially reduces tau-induced neurodegeneration in both the fly brain and retina. The issue of causality has previously been unresolved, although important studies have documented aberrant neuronal cell-cycle markers in human tauopathies and, more recently, in a mouse tauopathy model. The data also provide in vivo support for key experiments in cell-culture systems that have demonstrated cell cycle-dependent apoptosis in a variety of neurotoxic paradigms. Previous reports have implicated cell-cycle activation in rodent models of stroke and head trauma, although these studies have largely relied upon pharmacologic inhibition of the cell cycle by Cdk inhibitor drugs. Cdk inhibitors target several non-cell-cycle kinase targets, including GSK-3 and Cdk5, that have also been implicated in cell survival and tau phosphorylation. Indeed, while Cdk inhibitors were recently shown to be neuroprotective in a toxic mouse model of PD, this effect was found to be more attributable to inhibition of Cdk5 than to inhibition of cell cycle-related kinases (Khurana, 2006).
In this study, PCNA-, PH3-, and TUNEL-positive neurons were demonstrated in the tauopathy model and it was concluded that cell-cycle activation and apoptosis accompanied neurodegeneration. While expression of PCNA and PH3 have been described in processes other than cell division (DNA repair and immediate early gene responses, respectively), the genetic data implicating multiple components of the cell cycle in tau-induced neurodegeneration strongly support a cell-cycle role in this model. While TUNEL-positive cell death may not always be apoptotic, previous reports showing that antiapoptotic genes, including IAP-1, block tau-induced neurodegeneration in flies support a role for apoptosis in this model. The role of apoptosis in tauopathies and animal tauopathy models remains controversial, however. Recently, both apoptotic and nonapoptotic neurodegeneration were described in a mouse tauopathy model, and the possibility cannot be ruled out that nonapoptotic forms of cell death occur in the fly model also (Khurana, 2006).
The relationship between cell-cycle activation and tau phosphorylation has previously been unclear. Since Cdks are proline-directed kinases known to phosphorylate tau in vitro, cell-cycle activation could mediate neurodegeneration by directly phosphorylating tau. Indeed, several serine and threonine residues of tau are substrates for Cdks in vitro, and mitosis in cultured proliferating cells is associated with tau phosphorylation at these sites. Second, cell-cycle activation could be downstream of phosphorylation and directly lead to apoptosis in two plausible ways. First, forcing differentiated cells to enter a cell cycle could directly lead to apoptosis via an aborted attempt to replicate damaged DNA. Such a mechanism may be particularly relevant to postmitotic neurons that are known to have a limited capability for DNA repair. Alternatively, it is possible that cell-cycle mediators, including E2F1 and Cdk1, may subserve dual functions as direct mediators of neuronal apoptosis (Khurana, 2006).
The data support a role for cell-cycle activation downstream of tau phosphorylation and directly preceding apoptosis. First, cell-cycle markers often immunolocalized to areas characterized histologically by nuclear fragmentation and condensation, suggesting a late role in neurodegeneration. Second, cell-cycle modulation dramatically modifies tau-induced neurodegeneration without altering tau phosphorylation at disease-associated epitopes that can be generated by Cdks in vitro. In contrast, pseudophosphorylation of tau or reducing tau phosphorylation in a sgg mutant background directly increased and decreased cell-cycle activation, respectively. Third, cell-cycle modulation could still enhance toxicity of tauE14, a pseudophosphorylated construct in which all Ser-Pro and Thr-Pro target sites are mutated to glutamate. Fourth, double labeling of brains of tau-expressing flies for PH3 and tau phosphoepitopes revealed that >90% PH3-positive neurons were phosphoepitope positive, even for relatively restricted epitopes such as AT-180 (20% of all neurons). Finally, it was shown that cell-cycle activation, in the absence of transgenic tau, could directly lead to apoptosis of postmitotic neurons in vivo, supporting the possibility that cell-cycle activation could directly transduce tau-induced apoptosis (Khurana, 2006).
The mechanisms through which cell cycle becomes activated in tauopathies have not been defined. Markers that could represent aberrant mitogenic signaling are aberrantly expressed in these diseases, including markers of MAP kinase activation, classic oncogenic pathways such as Src and c-Myc, and TOR activation. However, the expression of isolated markers, while interesting, establishes neither the importance of a particular pathway as a whole nor whether any of these pathways are able to reactivate cell cycle in postmitotic neurons or lead to neurodegeneration in vivo. In this study, it was shown that TOR activation occurs in the current tauopathy model, recapitulating a similar finding in AD, and is furthermore required for neurodegeneration. Since TOR-dependent enhancement of tau toxicity is blocked by concomitant cell-cycle inhibition, and ectopic TOR activation leads to neuronal cell-cycle activation and apoptosis in the adult fly brain, the data indicate that TOR signaling mediates tau-induced neurodegeneration via cell-cycle activation. The relationship between tau, TOR, and cell-cycle activation, however, may be complex. In the retina, for example, TOR activation in the absence of transgenic tau results in an enlarged eye, whereas tau expression results in a small, rough eye. It is plausible that these phenotypic differences may be related to tau-induced pathogenic events that occur upstream of TOR activation in the described model. Also, while the data strongly link TOR to cell-cycle activation in the model, other TOR-dependent mechanisms of neurotoxicity cannot be ruled out. For example, TOR activation could theoretically enhance neurodegeneration by inhibiting autophagy, although the rescue of tau toxicity by loss of S6k would argue against this possibility since S6k is an activator of autophagy in flies (Khurana, 2006).
Aging is a significant risk factor for tauopathies. Interestingly, TOR inhibition is known to prolong lifespan in Drosophila, and the data thus directly link an aging-related signal transduction pathway to tau-induced neurodegeneration. Furthermore, withdrawal of amino acids in vitro or starvation in vivo results in inhibition of TOR signaling, potentially offering a molecular mechanism for the neuroprotection reported in human studies by caloric restriction (Khurana, 2006).
In this study, the role of cell-cycle activation in tau-mediated neurodegeneration was investigated because aberrant expression of cell-cycle markers is best described for tauopathies and AD. For example, one comprehensive study found upregulation of cell-cycle markers in AD and in a cohort of sporadic and inherited tauopathies but not in other diseases including PD, Huntington's disease, amyotrophic lateral sclerosis (ALS), or multiinfarct dementia. However, others have reported cell-cycle marker upregulation in the context of spinal cord injury, ALS, and PD. In addition, neuronal expression of cell-cycle markers has been described in several cell-culture paradigms of neurotoxicity and in mouse models of ataxia, leading to the speculation that cell-cycle activation might be a universal mechanism for neurodegeneration, perhaps related to oxidative stress. In the current study, however, no evidence of cell-cycle activation was found in fly models of either Machado Joseph Disease (MJD) protein or Parkinson's disease, despite the presence of apoptotic neurodegeneration, and cell-cycle modulation did not modify MJD-induced neurodegeneration. These data implicate distinct mechanisms for neurodegeneration in different neurodegenerative diseases, consistent with recent findings that forward genetic screen modifiers differ between polyglutamine-, synuclein-, and tau-induced neurodegeneration (Khurana, 2006).
In summary, the results indicate a common effector pathway and potentially common therapeutic strategies for cancer and tauopathy, two major causes of age-related morbidity and mortality. The TOR signaling pathway, a known regulator of lifespan, was delineated as a required mediator of tau-dependent neurodegeneration in vivo. The results provide a rationale for the assessment of TOR and cell-cycle inhibitors as potential therapeutic strategies in tauopathies and AD. (Khurana, 2006).
To survive starvation and other forms of stress, eukaryotic cells undergo a lysosomal process of cytoplasmic degradation known as autophagy. Autophagy has been implicated in a number of cellular and developmental processes, including cell growth control and programmed cell death. However, direct evidence of a causal role for autophagy in these processes is lacking, due in part to the pleiotropic effects of signaling molecules such as TOR that regulate autophagy. This study circumvents this difficulty by directly manipulating autophagy rates in Drosophila through the autophagy-specific protein kinase Atg1 (Atg signifies an autophagy-related gene). Overexpression of Atg1 is sufficient to induce high levels of autophagy, the first such demonstration among wild type Atg proteins. In contrast to findings in yeast, induction of autophagy by Atg1 is dependent on its kinase activity. Cells with high levels of Atg1-induced autophagy are rapidly eliminated, demonstrating that autophagy is capable of inducing cell death. However, this cell death is caspase dependent and displays DNA fragmentation, suggesting that autophagy represents an alternative induction of apoptosis, rather than a distinct form of cell death. In addition, this study demonstrates that Atg1-induced autophagy strongly inhibits cell growth, and that Atg1 mutant cells have a relative growth advantage under conditions of reduced TOR signaling. Finally, this study shows that Atg1 expression results in negative feedback on the activity of TOR itself. These results reveal a central role for Atg1 in mounting a coordinated autophagic response, and demonstrate that autophagy has the capacity to induce cell death. Furthermore, this work identifies autophagy as a critical mechanism by which inhibition of TOR signaling leads to reduced cell growth (Scott, 2007).
Under starvation conditions, eukaryotic cells recover nutrients via autophagy, a lysosome-mediated process of bulk cytoplasmic degradation. Through autophagy, long-lived proteins, organelles, and other components of the cytoplasm are non-selectively engulfed within specialized double-membraned vesicles known as autophagosomes. Subsequent fusion of the outer autophagosomal membrane with the lysosome results in a structure known as the autolysosome, in which the inner membrane and its cytoplasmic cargo are degraded. Breakdown products released from the autolysosome supply the cell with an internal source of nutrients that can support essential metabolic processes during starvation. Approximately 20 ATG (autophagy-related) genes specifically required for autophagy have been discovered in Saccharomyces cerevisiae, and many of these genes have functional homologs in metazoans (Scott, 2007 and references therein).
In addition to survival of starvation, autophagy has been implicated in many aspects of health and development, including aging, programmed cell death, pathogenic infection, stress responses, neurodegenerative and muscle disorders, cellular remodeling, cancer, and cell growth. As in many of these processes, evidence for a role of autophagy in cell growth control is largely correlative. Autophagy is promoted by several tumor suppressor genes including PTEN, TSC1, TSC2, and beclin 1, and is inhibited by growth-promoting pathways such as type I PI3K and target of rapamycin (TOR) signaling. A variety of conditions that stimulate cell growth, such as growth factor addition, partial hepatectomy, and refeeding after starvation, also inhibit autophagy, while growth suppressive signals, such as contact inhibition and substrate detachment, induce autophagy. Together, these correlative findings are consistent with a model in which the catabolic effects of autophagy act as a brake on cell growth during development. However, this issue is complicated by the pleiotropic nature of the signaling pathways that regulate autophagy. For example, in addition to inhibiting autophagy, the TOR pathway controls cell metabolism and biosynthesis by promoting ribosome biogenesis, protein synthesis and nutrient uptake. The relevant contribution of each of these downstream effector pathways to net cell growth is poorly understood (Scott, 2007).
Autophagy is also known to be involved in programmed cell death, and distinguishes Type II (autophagic) from Type I (apoptotic) cell death. Autophagic cell death is characterized by an abundance of autophagosomes and autolysosomes in the dying cell and differs from apoptotic cell death in that dying cells are degraded by their own lysosomal enzymes, rather than by phagocytosis. It has been difficult, however, to establish whether autophagy plays a causal role in Type II cell death or represents a failed attempt at cell survival in cells undergoing programmed cell death. Death signals often induce features of both apoptosis and autophagy, and mutations that disrupt autophagy have been shown to suppress cell death in some cases, and hasten it in others. Thus, as in the case for cell growth, much of the support for a direct role for autophagy in cell death rests on correlative evidence (Scott, 2007).
One approach to addressing the potential role of autophagy in functions such as cell death and growth would be to induce autophagy directly, independent of the signaling pathways that normally control it. In yeast, multiple signaling pathways, including TOR, AMPK and Ras/PKA, converge on the Ser-Thr kinase Atg1 to regulate autophagy. In addition, Atg1 interacts with multiple components of the autophagic machinery, through direct association, phosphorylation, and/or through effects on intracellular localization). Thus, Atg1 may represent a nodal point for controlling multiple steps in the autophagic process in response to various inductive cues (Scott, 2007).
Interestingly, the role of Atg1 kinase activity in yeast is the subject of some debate, and studies from different groups using ATP analog-sensitive and kinase-defective Atg1 mutants have reached conflicting conclusions. One study reported that Atg1 kinase activity is required for the autophagy-related biosynthetic cytoplasm to vacuole targeting (CVT) pathway but not for autophagy, and concluded that Atg1 plays a structural role in autophagy. However, other studies using similar reagents found a requirement for Atg1 kinase activity in both CVT and autophagy (Scott, 2007 and references therein).
It has been shown that the Drosophila homolog of Atg1 is required for autophagy in the larval fat body, an organ analogous to the vertebrate liver with roles in nutrient storage and mobilization. This study investigated the effects of Drosophila Atg1 loss of function and overexpression on autophagy induction, cell growth control, and cell death. The findings indicate that Atg1 expression is sufficient to effect a full autophagic response, resulting in a marked inhibition of cell growth and a rapid induction of apoptotic cell death (Scott, 2007).
Delivery of cytoplasmic components to the lysosome through autophagy involves multiple distinct steps including nucleation, expansion and closure of the autophagosome, and its subsequent fusion with the lysosome. These membrane trafficking events require the recruitment and subsequent retrieval of a large number of autophagy-specific Atg proteins, as well as general factors involved in vesicle trafficking. Given this complexity, the finding that overexpression of a single Atg protein is sufficient to accomplish all essential steps in this process is striking. As autophagy occurs constitutively at a basal rate in most eukaryotic cells, acceleration of a single rate-limiting step by overexpression of Atg1 may be sufficient to increase the overall rate of autophagy. Alternatively, the interaction of Atg1 with multiple Atg proteins suggests that Atg1 may function at multiple steps in the autophagic process. Identification of the relevant in vivo substrates of Atg1 will help to clarify this issue. A recent yeast proteomic microarray study identified a number of in vitro substrates of Atg1, including Atg8 and Atg18, which function in autophagosomal expansion and retrieval of components of the autophagic machinery, respectively, as well as general factors involved in vesicle transport and vacuolar function (Scott, 2007).
The role of the kinase activity of yeast Atg1 in regulating autophagy is the matter of some debate, since different groups have drawn conflicting conclusions. A consensus view, however, may be that autophagy and the CVT pathway require different levels of Atg1 kinase activity, or that Atg1 has different substrates under differing nutrient conditions. In higher eukaryotes, the role of Atg1 kinase activity may differ from that in yeast, as the CVT pathway has not been observed in metazoan cells. Consistent with the findings of Ohsumi and colleagues, however, the current results indicate that the kinase domain of Drosophila Atg1 is required for autophagy, since the kinase-inactive Atg1K38Q mutant failed to restore starvation-induced autophagy to Atg1 mutants, and was unable to induce autophagy when overexpressed. However, the partial reduction in size and TOR activity observed upon expression of Atg1K38Q, as well as its partial rescue of Atg1 mutants, indicate that Atg1 may have other, kinase-independent functions beyond regulating autophagy (Scott, 2007).
The mechanisms by which upstream signaling pathways regulate Atg1 are also likely to differ between yeast and multicellular organisms; essential components of the TOR-regulated Atg1 complex such as Atg13 and Atg17 are not readily identifiable in animal genomes. Nonetheless, the current results are consistent with a similar negative regulation of Atg1 by TOR, since activation of TOR signaling by Rheb overexpression or Tsc2 mutation suppresses the ability of Atg1 to induce autophagy. Increased Atg1 activity in response to loss of TOR signaling is likely to be critical for cell homeostasis and survival, since animals doubly mutant for Atg1 and Tor show a synthetic embryonic lethal phenotype. In addition, the results demonstrate an unexpected mode of signaling from Atg1 to TOR, in which increased Atg1 levels lead to downregulation of TOR kinase activity. It is unclear whether this reflects a direct effect of Atg1 on the activity of TOR or an upstream regulator, or an indirect consequence of the high level of autophagy in these cells, perhaps through increased turnover of TOR signaling components. The finding that a pool of TOR protein resides on intracellular vesicles and that TOR signaling is reduced in endocytic mutants suggests that TOR activity may respond to rates of vesicular trafficking, such as autophagy. Regardless of mechanism, these results suggest the existence of a self-reinforcing feedback loop, whereby increased Atg1 levels lead to downregulation of TOR activity, resulting in further activation of Atg1. In contrast, induction of autophagy in response to loss of TOR signaling is dampened by the resultant inactivation of S6K, which is required for normal autophagy. Thus TOR-mediated regulation of autophagy involves both positive and negative feedback (Scott, 2007).
Autophagy is a catabolic process that inversely correlates with cell growth, suggesting that increased levels of autophagy observed in growth-restricted cells may contribute to their reduced growth rate. The results presented here provide genetic support for this model. The data indicate that clones of autophagy-defective cells have a growth advantage over wild type cells under physiological conditions that normally induce autophagy, including starvation and rapamycin treatment. These results are confirmed by the marked size reduction of cells overexpressing Atg1, further supporting the role of autophagy as a negative effector of growth in TOR signaling. Thus, autophagy is partly responsible for the growth restriction resulting from physiological inhibition of TOR signaling, and is capable of inhibiting growth independent of TOR signaling (Scott, 2007).
In contrast to the growth advantage observed in autophagy defective cells, previous studies using yeast or cultured mammalian cells reported that inhibition of autophagy results in rapid cell death in response to starvation. This difference is likely due to the mosaic nature of the current experiments, in which clones of Atg1 mutant cells are surrounded by autophagy-competent wild type cells, and may in effect parasitize nutrients liberated through the autophagic activity of their neighbors. This is likely to be particularly evident in fat body cells, which are specialized for nutrient storage and mobilization. It is noted that this genetic mosaicism is similar to the situation facing tumor cells within wild type tissues. Thus, the increased growth capacity resulting from disruption of autophagy may contribute to the tumorigenicity of cells mutant for tumor suppressors such as PTEN, TSC1 & 2, beclin 1/Atg6 and possibly LC3/Atg8 and Atg7 (Scott, 2007 and references therein).
Whereas autophagy had an inhibitory effect on cell growth under physiological conditions, this was not the case in cells with severely disrupted TOR signaling, such as in cells with null mutations in Pdk1 Tor, despite the strong correlation of reduced cell growth and increased autophagy in these cells. Disruption of autophagy had no effect on the growth of Pdk1 null cells, and actually led to a further decrease in cell size of Tor mutants. In the complete absence of TOR signaling, nutrient uptake is severely curtailed, and under these extreme conditions the metabolic benefits of nutrients liberated by autophagy may outweigh its potential growth-inhibitory catabolic effect. It is concluded that autophagy has context-dependent effects on cell growth, providing for a rudimentary level of metabolism and growth under conditions of severe nutrient deprivation, acting as a net inhibitor of growth under conditions of reduced TOR signaling, and strongly inhibiting cell growth when induced to high levels. These findings add autophagy to the growing list of effector pathways and cellular processes through which TOR signaling controls cell growth, including translation, transcription, nutrient import and endocytosis (Scott, 2007).
Previous studies in a number of experimental systems indicate that the role of autophagy in cell death is also likely to be context-dependent. For example, autophagy has been found to protect against cell death in cases of growth factor withdrawal, starvation, and neurodegeneration, but to be required for some cases of autophagic cell death. Thus, observations of autophagic structures in dying cells are equally consistent with a causal, neutral or even inhibitory role of autophagy in cell death. The ability to induce autophagy through Atg1 overexpression has enabled a direct test of the potential role of autophagy in promoting cell death. The results indicate that induction of autophagy is sufficient to induce cell death. It was found that death resulting from Atg1-induced autophagy is suppressed by caspase inhibition and is associated with caspase activation, DNA fragmentation, and cytoskeletal disruption, suggesting that high levels of autophagy result in apoptotic cell death. The connection between apoptosis and autophagy is further supported by the recent demonstration that overexpression of the anti-apoptotic protein Bcl-2 can inhibit autophagy by interacting with the Atg6 homolog Beclin 1. Mutant versions of Beclin 1 that are unable to bind Bcl-2 stimulate autophagy and promote cell death, similar to the effects of Atg1 (Scott, 2007).
By what mechanisms might autophagy lead to cell death? The observation that starvation-induced autophagy is reversible and does not normally result in cell elimination suggests that starvation conditions may be protective against autophagy-induced death. Induction of autophagy is tolerated or even beneficial in cells with reduced biosynthetic activity, but may be detrimental in cells whose resources are devoted to continued growth. Self-limiting mechanisms also serve to prevent starvation-induced autophagy from proceeding at continuously high levels. During autophagic cell death, alternate activation of the autophagic machinery may circumvent these feedback mechanisms, resulting in high levels of sustained autophagy that are destructive to the cell. In addition, the level of Atg1 gene expression may be critical, since it is not upregulated under starvation conditions (Kirisako, 1999), but its levels peak at the beginning of the Drosophila pupal stage, when autophagic cell death is induced. Autophagic cell death may also result from the selective degradation of specific survival-promoting or death-inhibiting factors. In this regard, it was recently shown that the caspase-inhibitor zVAD results in targeted autophagic degradation of catalase, leading to accumulation of radical oxygen species and death (Scott, 2007).
In summary, these results demonstrate that constitutive induction of autophagy inhibits cell growth and leads to cell death, highlighting the importance of physiological mechanisms that restrain this process. In addition, the finding that Atg1 expression is sufficient to induce autophagy provides a new tool for experimental or therapeutic manipulation of autophagy. Compounds that target the kinase activity of Atg1 may lead to novel therapies for the wide range of diseases linked to autophagy (Scott, 2007).
Degradation of cytoplasmic components by autophagy requires the class III phosphatidylinositol 3 [PI(3)]-kinase Vps34, but the mechanisms by which this kinase and its lipid product PI(3) phosphate (PI(3)P) promote autophagy are unclear. In mammalian cells, Vps34, with the proautophagic tumor suppressors Beclin1/Atg6, Bif-1, and UVRAG, forms a multiprotein complex that initiates autophagosome formation. Distinct Vps34 complexes also regulate endocytic processes that are critical for late-stage autophagosome-lysosome fusion. In contrast, Vps34 may also transduce activating nutrient signals to mammalian target of rapamycin (TOR), a negative regulator of autophagy. To determine potential in vivo functions of Vps34, mutations were generated in the single Drosophila Vps34 orthologue (Phosphotidylinositol 3 kinase 59F), causing cell-autonomous disruption of autophagosome/autolysosome formation in larval fat body cells. Endocytosis is also disrupted in Vps34-/- animals, but this does not account for their autophagy defect. Unexpectedly, TOR signaling is unaffected in Vps34 mutants, indicating that Vps34 does not act upstream of TOR in this system. Instead, TOR/Atg1 signaling regulates the starvation-induced recruitment of PI(3)P to nascent autophagosomes. These results suggest that Vps34 is regulated by TOR-dependent nutrient signals directly at sites of autophagosome formation (Juhász, 2008).
Engulfment of cytoplasmic material into specialized double-membrane vesicles known as autophagosomes is the defining feature of a process referred to as macroautophagy or simply autophagy. Subsequent fusion of autophagosomes with the endolysosomal network leads to hydrolytic degradation of the sequestered material. This process provides eukaryotic cells with a mechanism for cytoplasmic renewal by which they can rid themselves of defective organelles and protein complexes. In addition, nonselective autophagy can be induced to high levels by starvation, providing an internal source of nutrients on which cells can survive extended periods of nutrient deprivation. Conversely, under some circumstances autophagy may be used as a killing mechanism, acting as an alternative or augmentation to apoptotic cell death. As autophagy has been implicated in several physiological and pathological conditions, including neurodegeneration, tumorigenesis, and aging, better understanding of the molecular mechanisms controlling autophagy and identification of pharmacological regulators of this process are important goals (Juhász, 2008).
Wortmannin and 3-methyladenine are well established inhibitors of autophagy. These compounds are broad-spectrum phosphatidylinositol 3 [PI(3)]-kinase inhibitors that disrupt autophagy by inhibiting Vps34, the enzymatic component of a multiprotein complex which also includes Vps15, Beclin1/Atg6, UVRAG, and Bif-1 in mammals and Vps15, Atg6, and Atg14 in yeast. Localized production of PI(3) phosphate (PI(3)P) by Vps34 can act to recruit proteins containing FYVE and PX domains to specific membrane compartment. In yeast, this Vps34 complex is critical for recruiting autophagy-related (Atg) proteins to the preautophagosomal structure, the yeast-specific site of autophagosome formation. The role of PI(3)P in autophagosome biogenesis is less well understood in higher eukaryotes, and whether it functions at the autophagosomal, the donor, or another membrane has not been determined (Juhász, 2008).
Vps34 is also required more broadly for several vesicular trafficking processes that may have indirect impacts on autophagy. These include sorting of hydrolytic enzymes to the lysosome/vacuole and early steps in the endocytic pathway. In mammalian cells, autophagosomes have been shown to fuse with early or late endosomes before fusion with lysosomes, resulting in intermediate structures known as amphisomes. Recently, mutations in components of the endosomal sorting complex required for transport (ESCRT) complex, which is required for the transition from early to late (multivesicular) endosomes, have been shown to block autophagy by inhibiting autophagosome-endosome fusion. Thus, the effect of PI(3)-kinase inhibitors on autophagy may be due, in part, to these more general trafficking functions of Vps34 (Juhász, 2008).
Recent work has shown that Vps34 can also function in a nutrient-sensing pathway upstream of the target of rapamycin (TOR) in several mammalian cell lines. Disruption of Vps34 activity with blocking antibodies or siRNA was found to inhibit activation of TOR by insulin, amino acids, and glucose. As TOR signaling inhibits autophagy, these findings are at odds with the conserved role of Vps34 in promoting autophagy under starvation conditions, suggesting that distinct complexes or pools of Vps34 may be subject to different modes of regulation (Juhász, 2008).
This paper addresses how these multiple potential roles of Vps34 are coordinated to regulate autophagy in Drosophila. The findings suggest that despite a critical role for Vps34 in endocytic uptake and recycling, its primary function in autophagy in vivo is limited to its direct role at the nascent autophagosome. Vps34 is not required for TOR activity in this system, and starvation results in a TOR/Atg1-dependent recruitment of Vps34 activity to the autophagosomal membrane (Juhász, 2008).
Previous work in mammalian and yeast systems has identified a wide range of vesicle trafficking processes regulated by Vps34, including autophagy, endocytosis, endosome maturation, and both anterograde and retrograde trafficking between the Golgi and lysosome. The current findings indicate that despite these activities, the in vivo role of Vps34 in autophagy is largely limited to its function at the autophagosome. Although fluid-phase endocytosis, endocytic recycling of Notch, and trafficking of lysosomal proteins are disrupted by mutation of Vps34, the results suggest that events subsequent to autophagosome formation, including fusion between autophagosomes and endosomes or lysosomes and subsequent lysosomal degradation, are not rate limiting in the absence of Vps34. Why does endocytic disruption lead to autophagosome fusion defects in ESCRT mutants but not in Vps34 mutants? Accumulation of endocytic tracer at the periphery of Vps34 mutant cells suggests that Vps34 functions at an early step of endocytosis, and apparently this event, as well as normal endocytic flux is not essential for fusion of autophagosomes with elements of the endosomal-lysosomal compartment. Interestingly, the accumulation of autophagosomes in ESCRT/Vps34 double mutants indicates that loss of Vps34 does not completely prevent autophagosome formation. Similarly, the lack of autophagosome accumulation in Vps34 single mutants indicates that ESCRT complexes are at least partially functional in the absence of Vps34. Thus, PI(3)P may not be absolutely essential for these processes, or perhaps sufficient levels of PI(3)P are generated independently of Vps34 by the class II PI(3) kinase or by PI(3,4)P or PI(3,5)P phosphatases. Since ESCRT components are required for multivesicular body formation but not autophagy in yeast, it will be interesting to determine whether the requirement for ESCRT complexes in autophagy in higher eukaryotes reflects their role in multivesicular body formation or an alternate function (Juhász, 2008).
The cellular compartment in which Vps34 acts to promote autophagy and the mechanisms by which it is regulated by nutrient signals have remained unresolved. In mammalian cells, Beclin1/Atg6 has been reported to localize to the trans-Golgi network, ER, and mitochondria. It was recently shown that Beclin1-Vps34 complexes can be inhibited by the antiapoptotic factor Bcl-2 in a nutrient-dependent manner. Bcl-2 mutants that are targeted to the ER, but not to mitochondria, retain their capacity to inhibit starvation-induced autophagy, suggesting that the ER is an important site of Beclin1-Vps34 regulation. However, it is unknown how these organelles contribute to the formation of autophagosomes, and recent studies suggest that rather than budding off a preexisting compartment, the autophagosomal membrane is likely to form de novo from small lipid transport vesicles or lipoprotein complexes. The finding that myc-2xFYVE is recruited to GFP-Atg8a-positive structures under starvation conditions indicates that Vps34 activity is targeted directly to autophagosomes in a TOR/Atg1-dependent manner. Although these results do not distinguish between a role for TOR/Atg1 signaling in regulating Vps34 activity versus providing a platform on which Vps34 complexes can assemble, together with these previous studies they indicate that Vps34 is likely to promote autophagy by different mechanisms from multiple cellular locations (Juhász, 2008).
How the TOR signaling pathway senses intracellular levels of nutrients, such as amino acids, has been poorly understood despite considerable work in yeast, mammalian, and other model systems. The recent identification of Vps34 as a transducer of this signal in mammalian cells thus represents a significant new insight into this issue. However, further work is necessary to determine the extent to which this mechanism is generally conserved, since starvation appears to have opposing effects on Vps34 activity in different cell types. The results presented in this study fail to support this model in Drosophila; mutation of Vps34 does not appear to influence TOR-dependent phenotypes nor to disrupt TOR-dependent signaling. This may reflect a fundamental difference in signaling mechanisms between the fly and mammalian systems. The makeup of Vps34 complexes has diverged significantly between yeast and metazoans, and perhaps components of this complex, such as Ambra1, that appear to be unique to vertebrates may confer functions not found in flies. It is also possible that production of PI(3)P by Vps34-independent mechanisms is more efficient in D. melanogaster than in mammalian cells and, thus, a role for Vps34 in TOR signaling may be obscured by these other sources. The continued ESCRT function and basal level of autophagy in Vps34 null mutants are consistent with this possibility. Alternatively, the current findings may reflect important differences between the roles of TOR and Vps34 in vivo versus in cultured cells as well as the experimental paradigms of these systems. For example, although complete starvation is commonly used to inactivate TOR in cell culture studies, such experiments may not accurately mimic physiologically relevant events, given the inherent capacity of intact organisms to buffer changes in nutrient levels. Additional studies in an in vivo mammalian system will be helpful to clarify these issues (Juhász, 2008).
The TOR kinases are conserved negative regulators of autophagy in response to nutrient conditions, but the signaling mechanisms are poorly understood. This study describes a complex containing the protein kinase Atg1 and the phosphoprotein Atg13 that functions as a critical component of this regulation in Drosophila. Knockout of Atg1 or Atg13 results in a similar, selective defect in autophagy in response to TOR inactivation. Atg1 physically interacts with TOR and Atg13 in vivo, and both Atg1 and Atg13 are phosphorylated in a nutrient-, TOR- and Atg1 kinase-dependent manner. In contrast to yeast, phosphorylation of Atg13 is greatest under autophagic conditions and does not preclude Atg1-Atg13 association. Atg13 stimulates both the autophagic activity of Atg1 and its inhibition of cell growth and TOR signaling, in part by disrupting the normal trafficking of TOR. In contrast to the effects of normal Atg13 levels, increased expression of Atg13 inhibits autophagosome expansion and recruitment of Atg8/LC3, potentially by decreasing the stability of Atg1 and facilitating its inhibitory phosphorylation by TOR. Atg1/Atg13 complexes thus function at multiple levels to mediate and adjust nutrient-dependent autophagic signaling (Chang, 2009).
The remarkable conservation of autophagy-related proteins and processes between yeast and higher eukaryotes has contributed to a rapid advance in understanding of this process in multicellular animals. The data presented in this study extend this conservation to the mechanisms of autophagy regulation by nutrient-dependent TOR signaling. As in yeast, Drosophila Atg13 forms a complex with Atg1, stimulates its activity and is highly phosphorylated in a nutrient and TOR-dependent manner. Although the relevant substrates of Atg1 remain to be identified, these results suggest that this fundamental pathway from nutrient signal to autophagic induction has been largely retained between yeast and metazoans. This regulatory link between TOR and Atg1/Atg13 thus represents one of the most highly conserved TOR outputs described to date. Despite this conservation, critical differences were identified in the behaviors of these proteins, which likely stem in part from differences in the physiology of lower vs. higher eukaryotes. An essential role in autophagy has been reported for human Atg13 using RNAi-mediated knockdown in cultured cell lines, and interactions were described between Atg13 and Ulk1 and Ulk2 similar to those reported in this study. Others have also described related interactions between mammalian Atg13 and Ulk kinases. Together, these studies point to a model whereby Atg13 is phosphorylated and interacts with Atg1 under both fed and starved conditions in metazoans, in contrast to the growth-dependent phosphorylation and dissociation of Atg13/Atg1 observed in yeast. Although the greater nutrient buffering capacity of multi- vs. unicellular animals probably contributes to these differences, the requirement for significant basal rates of autophagy in the maintenance of large, long-lived metazoan cells may dictate that the Atg1-Atg13 complex remain partially active even under fed conditions. Furthermore, although Atg1 can clearly associate with phosphorylated Atg13, dephosphorylation of TOR-dependent sites on Atg13 may be masked by increased phosphorylation by Atg1, but may nonetheless contribute to regulation of Atg1-Atg13 interaction or activity. The observation that Atg1 kinase activity plays a role under both fed and starved conditions in supporting hyperphosphorylation of Atg13 suggests that rather than switching Atg1 kinase between off and on states, nutrient signals may instead affect its substrate specificity or accessibility (Chang, 2009).
Metazoan Atg1/Atg13 complexes have also accrued additional regulatory mechanisms that have not been described in yeast, including negative feedback from Atg1 to TOR, phosphorylation of Atg13 by Atg1, and nutrient-dependent effects on the stability of these proteins. These additional layers of regulation are in keeping with the elaboration of intracellular signaling pathways in metazoans. The observation that Atg13 phosphorylation is both TOR- and Atg1-dependent under fed conditions suggests a model whereby phosphorylation by one of these kinases may serve as a priming event for the other. In addition, the role of Atg1-dependent phosphorylation of Atg13 under starvation conditions remains an important question. Whereas the Atg1-independent localization of Atg13 to autophagosomes and its activation of Atg1 indicate that Atg13 functions upstream of Atg1, the finding that Atg13 acts as a substrate for Atg1-dependent phosphorylation raises the possibility that Atg13 may also act to transduce signals downstream of Atg1. Identification of additional Atg1 substrates and Atg13 interacting proteins may help clarify this issue (Chang, 2009).
Given its positive role in autophagy induction, the ability of Atg13 to inhibit autophagy when overexpressed was unexpected. This may in part reflect a dominant negative effect of Atg13 overexpression causing titration of Atg1 complexes or competition with other Atg1 substrates. However, the observation that Atg13 stimulates TOR-dependent phosphorylation of Atg1 and that Atg1 levels increase in Atg13 mutant cells suggests that Atg13 has both positive and negative roles in autophagy induction. These opposing activities of Atg13 are reminiscent of the mTOR complex I component Raptor, which plays an essential role in TOR signaling yet also inhibits TOR activity under starvation conditions. The results suggest that Atg13 may play an analogous role, switching between states of promoting or inhibiting autophagy, thereby sharpening the response to changes in nutrient conditions. These findings suggest that the relative ratio of Atg13 to Atg1 or to other components of the complex may play an important role in dictating its activity. In this regard, the differential regulation of Atg1 and Atg13 levels by TOR could provide an additional mechanism whereby TOR signaling can influence autophagic activity. Whereas the autophagy-defective phenotypes of Atg1 and Atg13 mutants reveals the dominant positive roles of these genes, disruption of other putative components of this complex leads to autophagy induction, suggesting some components may play primarily negative roles (Chang, 2009).
In conclusion, the results demonstrate that Atg1/Atg13 complexes play an essential, conserved role in promoting autophagy in response to TOR inactivation, with additional regulatory functions unique to metazoans. Further insight into the regulation of this complex and identification of its targets may lead to additional means of manipulating autophagy rates for therapeutic purposes (Chang, 2009).
The TOR pathway mediates nutrient-responsive regulation of cell growth and metabolism in animals. TOR Complex 1 activity depends, among other things, on amino acid availability. MAP4K3 was recently implicated in amino-acid signaling in cell culture. This study reports the physiological characterization of MAP4K3 mutant flies. Flies lacking MAP4K3 have reduced TORC1 activity detected by phosphorylation of S6K and 4EBP. Furthermore MAP4K3 mutants display phenotypes characteristic of low TORC1 activity and low nutrient availability, such as reduced growth rate, small body size, and low lipid reserves. The differences between control and MAP4K3 mutant animals diminish when animals are reared in low-nutrient conditions, suggesting that the ability of TOR to sense amino acids is most important when nutrients are abundant. Lastly, physical interaction is shown between MAP4K3 and the Rag GTPases raising the possibility they might be acting in one signaling pathway (Bryk, 2010).
The multiprotein complex TORC1, containing TOR kinase, is a central regulator of cellular growth and metabolism in animals. It is activated by a number of inputs relating to cellular energy and nutrient status. These include insulin, glucose, cellular energy levels and amino acid availability. In response, TORC1 activates protein synthesis via a number of mechanisms including activation of the ribosomal S6 kinase (S6K), repression of the translational inhibitor 4E-BP, and promotion of ribosome biogenesis via myc. In particular since TORC1 is a master regulator of protein biosynthesis, its regulation by amino acids, the building blocks of proteins, likely constitutes an important regulatory feedback mechanism. Furthermore, the importance of amino acid signaling to TOR is highlighted by the observation that circulating amino acids are elevated in humans with obesity, where they have been shown to activate TORC1 activity and modulate glucose metabolism. Despite this, understanding of the molecular mechanism by which amino acids regulate TOR remains fragmentary (Bryk, 2010).
Three protein complexes have recently been implicated in the activation of TORC1 in response to amino acids. The human class III PI3K (phosphoinositide 3-kinase) hVps34 is activated by amino acids via a calcium dependent mechanism (Gulati, 2008). This leads to accumulation of phosphatidylinositol 3-phosphate (PI(3)P) in cells, which is thought to cause the recruitment of proteins recognizing PI(3)P to early endosomes, forming an intracellular signaling platform that leads to TORC1 activation. This feature of the pathway may be specific for vertebrates, as flies mutant for Vps34 have been reported to not have TORC1 signaling defects. Recently, two groups discovered that Rag GTPases mediate amino acid signaling to TORC1. The emerging picture is that amino acids change the GDP/GTP loading of the Rag GTPases, thereby stimulating the binding of Rag heterodimeric complexes to TORC1. This in turn causes TORC1 to change its intracellular localization, perhaps relocalizing it to vesicles containing the activator Rheb. This mechanism appears to be evolutionarily conserved from flies to humans (Bryk, 2010).
The third protein recently identified as a mediator of amino acid signaling to TOR is MAP4K3 (Findlay, 2007); in HeLa cells the kinase activity of MAP4K3 is activated in the presence of amino acids. In turn, MAP4K3 is required for TOR to phosphorylate its targets S6K and 4E-BP1 in response to amino acid sufficiency. The cell-culture data also suggest this mechanism is conserved from flies to humans as knockdown of Drosophila MAP4K3 causes a reduction in TOR activity (Bryk, 2010).
Although considerable progress has been made, important questions remain unanswered. The role of TORC1 activity in vivo has been well studied in flies and mice, but fundamental issues regarding the regulation of TOR by amino acids have thus far only been explored in vitro in cell culture. To assess the functional significance of the ability of TORC1 to sense amino acids in the organismal context, use has been made of a Drosophila mutant for MAP4K3 (CG7097). dMAP4K3 mutant flies have reduced TOR activity, detected by phosphorylation of TOR targets. dMAP4K3 mutants are viable but display physiological aberrations emblematic of animals starved of nutrients: MAP4K3 mutant flies have retarded growth, reduced size, and low lipid reserves. Both the biochemical results and the phenotypes indicate that MAP4K3 modulates, but is not absolutely required, for TOR activity in vivo. This is similar to what is observed with other modulators of the pathway, such as Melted. Unexpectedly, the function of MAP4K3 is most required when nutrient conditions are rich (Bryk, 2010).
Recent reports have shown that not all components identified in cell culture as regulators of TORC1 activity also affect TORC1 in vivo in an animal model. The purpose of this study was two-fold: (1) to analyze whether MAP4K3 regulates TORC1 activity in vivo in the fly, and (2) to study the physiological consequences for the organism when the ability of TORC1 to sense amino acids is impaired (Bryk, 2010).
Biochemical evidence is presented that TORC1 activity is reduced in MAP4K3 mutant animals, consistent with published cell-culture data showing that MAP4K3 is required for full TORC1 activation (Findlay, 2007). Furthermore, MAP4K3 mutants have defects typical of reduced TORC1 activity. They are delayed in their development due to a reduced rate of growth. They eventually pupate leading to adults of reduced size and their tissues are comprised of cells that are smaller than normal. Furthermore, MAP4K3 mutants have significantly reduced triglyceride stores compared to controls. These physiological effects are similar to the phenotypes observed with mutants for other regulators of TOR, such as Melted. Melted mutant flies are also 10% smaller than controls and are significantly leaner (Bryk, 2010).
As a whole, the MAP4K3- mutant phenotypes emulate the physiological effects observed when flies are grown on conditions of limiting food. When wildtype larvae are put on a low-nutrient diet, they are delayed in pupation and yield animals of small size that are lean. Thus loss of MAP4K3 activity phenocopies a reduced nutrient environment, consistent with MAP4K3 playing a role in the ability of animals to sense their nutrient conditions. This suggests the ability of TORC1 to sense amino acids is most important when nutrient conditions are rich, allowing animals to accelerate their growth accordingly. In contrast, on a low-nutrient diet, control and MAP4K3 mutant flies grow equally slowly consistent with TOR activity being low in both groups. This parallels nicely the results reported in cell culture by (Findlay, 2007): In the absence of amino acids, both control and MAP4K3 knockdown cells have low TOR activity whereas in the presence of amino acids, TOR is activated strongly in control cells but only weakly in MAP4K3 knockdown cells (Bryk, 2010).
Unexpectedly, it was found that MAP4K3 mutant animals are viable, although they have an elevated mortality rate compared to controls. This suggests that the amino acid sensing pathway might only modulate TORC1 activity. If TORC1 activity were completely blunted in MAP4K3 mutants, the animals would be dead, as is the case for TOR or Rheb mutants. Consistent with this, residual TORC1 activity was observed in MAP4K3 mutants, as detected by phosphorylation of the TORC1 targets S6K and 4EBP. This also parallels results from cell culture. The results presented in Findlay (2007) are obtained with cells starved of serum and consequently of insulin signaling. In the presence of insulin signaling, which resembles the physiological situation more closely, MAP4K3 mutant cells still retain residual TOR activity, similar to what was observed in vivo in this study. Consistent with these findings, it was observed that the Rheb expression is able to drive tissue growth also in the absence of MAP4K3 (Bryk, 2010).
Both MAP4K3 and the Rag GTPases have recently been shown to be required for amino acids to stimulate TORC1 activity. While studying dMAP4K3, it was noticed that dMAP4K3 binds physically to the Rag GTPases, suggesting they might act together as components of a single signaling pathway. This interaction is likely specific for several reasons: (1) no binding of MAP4K3 to another GTPase, Rheb, could be detected, (2) binding of MAP4K3 to the RagA/C complex was significantly stronger than binding of an unrelated HA-tagged protein, HA-medea (3) MAP4K3 bound FLAG-RagC significantly stronger than FLAG-RagA showing that MAP4K3 distinguishes between two Rag proteins and (4) binding of MAP4K3 to RagC depended on its GDP/GTP state (Bryk, 2010).
Further studies will be required to test whether this interaction is important for TORC1 to sense amino acids. The data suggest that MAP4K3 might be functioning upstream of the Rag GTPases, and not downstream since activated RagA does not require MAP4K3 to promote tissue growth in vivo. This raises the possibility that the Rag GTPases may be substrates for MAP4K3 phosphorylation. Indeed, RagC is phosphorylated in vivo in Kc167 cells on Ser388. If MAP4K3 were to phosphorylate RagC, this would provide a mechanism for regulation of the Rag GTPases, which to date is mysterious. Work in the near future should shed further light on this issue (Bryk, 2010).
In summary, this study has characterized the physiological function of MAP4K3 in Drosophila, and shown that it modulates TORC1 activity, tissue growth and lipid metabolism in the animal. Physical interaction data hints at a possible link between MAP4K3 and the Rag GTPases. The organismal function of amino acid sensing by TORC1 is mainly required to spur growth when nutrient conditions are rich (Bryk, 2010).
MAP4K3 is a conserved Ser/Thr kinase that has being found in connection with several signalling pathways, including the Imd, EGFR, TORC1 and JNK modules, in different organisms and experimental assays. This study analyzed the consequences of changing the levels of MAP4K3 expression in the development of the Drosophila wing, a convenient model system to characterize gene function during epithelial development. Using loss-of-function mutants and over-expression conditions it was found that MAP4K3 activity affects cell growth and viability in the Drosophila wing. These requirements are related to the modulation of the TORC1 and JNK signalling pathways, and are best detected when the larvae grow in a medium with low protein concentration (TORC1) or are exposed to irradiation (JNK). MAP4K3 was also shown to display strong genetic interactions with different components of the InR/Tor signalling pathway, and can interact directly with the GTPases RagA and RagC and with the multi-domain kinase Tor. It is suggested that MAP4K3 has two independent functions during wing development, one related to the activation of the JNK pathway in response to stress and other in the assembling or activation of the TORC1 complex, being critical to modulate cellular responses to changes in nutrient availability (Resnik-Docampo, 2011).
This study has characterised the consequences of changing the amount of MAP4K3, encoded by hppy, in the development of the wing disc, focussing on its relationships with the TORC1 signalling pathway. Previous data suggested that MAP4K3 might be related with a variety of signalling pathways, including EGFR, ImD, JNK and TOR. For these reasons, the advantages of the wing model was used to analyse hppy, as in this system changes in the level of signalling by a variety of pathways lead to pathway-specific phenotypes. A reduction of hppy expression in the wing, using interference RNA or loss-of-function alleles, did not uncover a critical requirement of the gene for embryonic or larval viability. In hppy mutant wings, only a weak reduction was found in wing size and cell size, compatible with a moderate reduction of TORC1 activity. It has been recently reported (Bryk, 2010) that the developmental delay caused by protein starvation is similar in wild type and hppy mutant larvae, suggesting that MAP4K3 is required in vivo to activate TOR and promote growth mostly when amino acid conditions are rich. In contract, this study found a significant requirement for the gene when hppy mutant larvae grow under starvation conditions. Thus, these flies still develop smaller wings than controls, indicating a functional requirement of hppy when the availability of proteins is reduced. This difference could be due to the parameters measured (developmental delay vs. cell and wing size) or to the remnants of hppy function in the alleles used in each experiment (Resnik-Docampo, 2011).
It was also found that, loss of hppy does not affect cell viability or JNK signalling, but that in a hppy loss-of-function genetic background the activation of JNK signalling in response to irradiation is reduced. Thus, the function of hppy might become significant mostly when the organism is challenged by stress signals induced for example by irradiation, indicating a role for the gene in the modulation of JNK signalling in vivo (Resnik-Docampo, 2011).
The increase in happyhour expression does have more dramatic consequences than its loss, causing a severe reduction in the size of the wing independently of environmental conditions. Wing size reduction is associated with both apoptosis and a smaller than normal cell size. The overall morphology and pattern of these wings is normal, with only a weak phenotype of extra-veins in the strongest combinations. Cell death induction and reduced cell size are the diagnostic phenotypes of increased JNK and reduced InR/Tor signalling, respectively. The same processes are affected by loss of MAP4K3 expression in the wing, and therefore, from this analysis it is concluded that MAP4K3 has the potential to activate cell death through the JNK signalling pathway, and also that it can interfere with some component/s of the InR/Tor cascade. The effects of loss- and gain of MAP4K3 on JNK activity are opposite, which is expected from a protein with kinase activity. In contrast both loss and gain of MAP4K3 seem to reduce the function of TORC1. It is likely that in this case MAP4K3 acts as part of a protein complex that can be made non-functional by changes in the stochiometry of its components. What seems clear is that the effects of MAP4K3 on JNK and TORC1 are exerted through independent mechanisms, because the contribution of cell death to the wing phenotype of MAP4K3 over-expression is very modest, and Tor reductions only lead to cell death when cells with different levels of Tor activity are confronted (Resnik-Docampo, 2011).
The phenotype of MAP4K3 over-expression is very sensitive to changes in the levels or activity of several members of the InR/Tor pathway. Thus, strong synergistic interactions were observed when Akt, raptor and Tor are reduced in the background of MAP4K3 over-expression, and the presence of the dominant-negative form TorTED in this background entirely eliminates the wing. Conversely, loss of hppy expression rescues the effects of TorTED expression. These results suggest that MAP4K3 could act at the level of TORC1. This possibility is compatible with the suppression by MAP4K3 over-expression of phenotypes caused by increased levels of InR/Tor signalling generated by lower than normal levels of PTEN and TSC1/2. In addition to genetic data in the wing, experiments in cell culture with both the fly and human MAP4K3 homologue proteins indicated that MAP4K3 is required to generate maximal activity of TORC1 in response to aminoacids. Therefore, it is suggested that although MAP4K3 is normally required to promote TORC1 signalling, when the protein is over-expressed the balance between TORC1 components required for its normal function in vivo is modified. This effect appears to depend exclusively on the kinase domain of MAP4K3, because the over-expression of this domain causes a strong reduction in wing and cell size. This sstudy has shown that MAP4K3 can interact with RagA, RagC and Tor in pull-down experiments in vitro, and therefore it is speculated that the excess of MAP4K3 alters the phosphorylation levels of TORC1 components and this leads to the assembly of inactive complexes. A similar mechanism might explain the dominant-negative effect of Tor, as it was suggested that Tor over-expression leads to the sequestering of TORC1 components in non-functional complexes. In summary, it is suggested that MAP4K3 normally potentiate TORC1 and JNK functions in response to environmental challenges, without being strictly required to generate some levels of TORC1 or JNK activity, and that MAP4K3 hyper-activity leads to high levels of JNK signalling and to reduced TORC1 function, in this case due to the formation of inactive TORC1 complexes (Resnik-Docampo, 2011).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.