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. 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).
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