InteractiveFly: GeneBrief

SNF1A/AMP-activated protein kinas: Biological Overview | References

LKB1 is mutated in both familial and spontaneous tumors, and acts as a master kinase that activates the PAR-1 polarity kinase and the adenosine 5'monophosphate-activated kinase (AMPK). This has led to the hypothesis that LKB1 acts as a tumor suppressor because it is required to maintain cell polarity and growth control through PAR-1 and AMPK, respectively. However, the genetic analysis of LKB1-AMPK signaling in vertebrates has been complicated by the existence of multiple redundant AMPK subunits. This study describes the identification of mutations in the single Drosophila AMPK catalytic subunit AMPKα. Surprisingly, ampkα mutant epithelial cells lose their polarity and overproliferate under energetic stress. LKB1 is required in vivo for AMPK activation, and lkb1 mutations cause similar energetic stress-dependent phenotypes to ampkα mutations. Furthermore, lkb1 phenotypes are rescued by a phosphomimetic version of AMPKα. Thus, LKB1 signals through AMPK to coordinate epithelial polarity and proliferation with cellular energy status, and this might underlie the tumor suppressor function of LKB1 (Mirouse, 2007).

LKB1 is a serine/threonine kinase that is mutated in most cases of Peutz-Jeghers syndrome, which is an autosomal dominant disorder in which patients develop benign hamartomas and a high frequency of malignant tumors (Alessi, 2006). Furthermore, LKB1 is also mutated in some sporadic cancers, such as 30% of lung adenocarcinomas, and the expression of the kinase is also down-regulated in a substantial proportion of breast cancers (Sanchez-Cespedes, 2002). In both cases, tumors associated with LKB1 mutations usually derive from epithelial tissue. LKB1 is a master kinase that can potentially activate several downstream kinases by phosphorylating a conserved threonine in their activation loops (Lizcano, 2004). Two of these kinases have been extensively characterized: PAR-1/microtubule affinity-regulating kinases (MARKs) and AMPK (AMP-activated protein kinase). PAR-1 regulates cell polarity in numerous cell types and organisms (see Drosophila Par-1). AMPK acts as a cellular energy sensor because it is activated by AMP, which accumulates when ATP levels are low (Kahn, 2005). AMPK then mediates the cellular response to energetic stress by activating energy-producing activities, while inhibiting energy-consuming ones, such as translation and proliferation. LKB1 regulates both cell polarity and cell growth and division in cell culture and in vivo. One hypothesis envisions LKB1 signaling mediated by PAR-1 regulating cell polarity, whereas LKB1 signaling through AMPK could control cell growth and proliferation. However, recent cell culture experiments suggest that AMPK also plays a role in the polarization of MDCK cells by promoting tight junction assembly (Zhang, 2006; Zheng, 2007). This study shows that LKB1 and AMPK are required to maintain epithelial polarity and integrity under energy-limiting conditions in Drosophila melanogaster. Therefore, these results provide a potential mechanism to coordinate the regulation of cell polarity and proliferation with energy conditions within a multicellular animal (Mirouse, 2007).

AMPK contains three protein subunits, α, β, and γ, which form a heterotrimer. The α subunit (AMPKα) encodes a highly conserved serine/threonine kinase, and the other subunits are regulatory. From a D. melanogaster forward genetic screen for mutants affecting larval neuronal dendrite development (Medina, 2006), several lethal mutations were identified in AMPKα. The ethylmethanesulfonate mutants, ampkα¹ and ampkα², contain a single amino acid change (S211L, completely conserved) and a premature stop codon (Q295 STOP), respectively, whereas ampkα³ has a 16-bp deletion creating a stop codon (Y141 STOP). All ampkα mutants, whether homozygous or in trans with a deletion covering the locus, displayed a completely penetrant and nearly identical phenotype, with greatly enlarged plasma membrane domains in dendrites, but not in axonal compartments. In addition, ampkα¹ and ampkα³ could be rescued to viability with either a chromosomal duplication carrying a wild-type ampkα gene, a wild-type AMPKα transgene, or a transgene that is tagged with the red fluorescent protein mCherry. The requirement for ampkα is cell autonomous because transgene expression within only neurons rescues the phenotype. Therefore, these mutations represent the first knockouts of the single AMPKα catalytic subunit in the Drosophla genome and allow the genetic analysis of AMPK function in vivo (Mirouse, 2007).

Although ampkα mutants display a strong phenotype in larval neuronal dendrites, no phenotype was observed in early larval lkb1 mutants, probably because of the large maternal contribution of this protein. To explore the relationship between AMPKα and LKB1 function without the confounding issues caused by the differing maternal contributions of each protein, follicle cells of the Drosophila ovary were examined. The follicle cells that surround the oocyte have a typical epithelial architecture with a highly polarized actin cytoskeleton in which the apical surface is marked by dense actin bundles in the apical microvilli, the lateral cortex is covered by a thin actin mesh, and the basal side contains a prominent network of parallel actin stress fibers. This polarized organization of actin typifies many epithelia, including the main mammalian tissue culture model for polarized epithelial cells, MDCK cells. No actin phenotypes were observed in ampkα³ mutant follicle cells using standard detection procedures. Because AMPK is maximally activated under low cellular energy levels, the influence of energy stress was tested by strongly reducing the availability of sugar in the Drosophila culture medium. Under these conditions, ampkα³ mutant cells display a strong actin phenotype. The density of basal stress fibers is strongly reduced, whereas the amount of apical F-actin increases. This phenotype is highly penetrant under these starvation conditions and is also observed with the two other alleles of ampkα (Mirouse, 2007).

Because this phenotype reflects a disruption of the apical–basal polarity of the actin cytoskeleton, polarity markers were examined within these cells. ampkα mutant clones induced in adult flies fed with high-sugar diets did not show any polarity phenotypes, which is consistent with the absence of an actin phenotype under these conditions. Under energetic starvation conditions, however, ampkα mutant cells show a fully penetrant loss of polarity. Apical markers, such as atypical PKC (aPKC) and Crumbs (Crb) lose their cortical localization completely and appear to be down-regulated, as do the lateral markers Discs large (Dlg) and Coracle (Cora). In contrast, Dystroglycan (Dg), which is normally enriched at the basal cortex, extends into the lateral domain, and occasionally even reaches the apical membrane. This suggests that the phenotype represents an expansion of the basal domain at the expense of the lateral and apical domains (Mirouse, 2007).

Although most aspects of apical–basal polarity are completely disrupted in ampkα mutant clones under energetic stress, E-cadherin (ECad) is usually still enriched at the adherens junctions, suggesting that the altered polarity is not a secondary consequence of a loss of intercellular adhesion. The subapical localization of Bazooka (Baz) with cadherin is also maintained in most cases. This indicates that Baz is not in a complex with aPKC in columnar follicle cells, but is instead associated with the adherens junctions (Mirouse, 2007).

A considerable proportion of ampkα mutant clones show a more severe phenotype, in which the cells round up and lose their epithelial organization to form multiple layers of cells. In these cases, Baz is now also absent from the cell cortex. Finally, larger mutant clones, particularly at the anterior or the posterior of the egg chamber, show a complete loss of epithelial organization and overproliferate to form small, tumorlike growths (Mirouse, 2007).

As one proposed function for AMPK is to sense and maintain cellular ATP levels, the polarity phenotype observed under starvation conditions could be caused by low cellular ATP concentrations. To test this hypothesis, cells were examined that were mutant for tenured (tend). Tend encodes a mitochondrial cytochrome oxidase subunit; therefore, mutants have reduced intracellular ATP concentrations to levels sufficient to maintain cell survival and growth, but not cell division. This cell cycle block is believed to require AMPK activation. In agreement with a role for Tend in cell cycle progression, tend clones bigger than four to six cells under were not observed energetic starvation conditions. In contrast to ampkα mutant cells, however, tend mutant cells showed no polarity defects, ruling out the possibility that the ampkα phenotype is a secondary effect of low ATP levels. The effect of specific nutrient starvation was also tested by feeding flies only glucose, but these conditions did not induce any polarity phenotypes in ampkα mutant cells. Thus, AMPKα is specifically required to maintain epithelial polarity and growth control under conditions of energetic stress (Mirouse, 2007).

Because the results indicate that ampkα plays a role in epithelial polarity, whether the localization of the protein itself is polarized was assessed. LKB1 localization was also examined, since it is a potential regulator of AMPK. Transgenic wild-type fusion proteins for both AMPKα and LKB1 rescue lethal null mutants to viability, and should therefore mimic the localizations of the endogenous proteins. LKB1-GFP is mainly found at the apical and lateral cortex of the follicle cells, and is absent from the basal domain. This basal exclusion is surprising, as cortical localization of LKB1 requires its membrane targeting by prenylation of a conserved CAAX motif. This suggests that the lipid composition of the basal domain is different from the rest of the plasma membrane and/or that LKB1 posttranslational modifications are asymmetrically controlled. In contrast, mCherry-AMPKα does not show any enrichment or asymmetric localization at the plasma membrane, and it is found distributed throughout the cytoplasm, but absent from the nucleus. The localization of LKB1 suggests that AMPK could be activated specifically at the apical and lateral cortices of the cells. To test this hypothesis, an antibody was used against the LKB1 phosphorylation site of AMPK (phospho-T184). The immunostaining is reduced to background levels in both ampkα and lkb1 mutant clones. This confirms the specificity of the antibody and indicates that LKB1 is the principle AMPK kinase in these cells. In wild-type cells, PhosphoT184-AMPK is found diffusely in the cytoplasm. The effect of AMPK on apical–basal polarity is therefore not related to a polarized distribution of the kinase or its localized activation by LKB1 (Mirouse, 2007).

Because LKB1 activates AMPK, it was asked if similar phenotypes could be observed in lkb1 mutant cells. lkb1 clones can lead to severe polarity defects in follicle cells in normally fed flies. However, these defects are observed only in large clones that are induced in the stem cells that give rise to the follicular epithelium, whereas small lkb1 mutant clones, which are induced after the formation of the epithelium, have no effect on follicle cell polarity or the organization of the actin cytoskeleton. This suggests LKB1 is required for the establishment of epithelial polarity in well-fed flies, but not for its maintenance, as is the case for PAR-1. In contrast, under conditions of glucose starvation, small lkb1 clones that were induced after the formation of the follicular epithelium show a fully penetrant polarity phenotype. Under these conditions, a loss of the polarized localization of Dlg, aPKC, Crb, and Cora was observed. However, Baz distribution is usually not affected by lkb1 loss of function. Dg extends laterally and occasionally localizes to the apical domain. The actin cytoskeleton is also disturbed, with more F-actin apically and a decreased density of stress fibers on the basal side. Finally, large lkb1 clones lose their epithelial organization completely and overproliferate to form small neoplasms. Thus, lkb1 mutant cells exhibit identical phenotypes to ampkα mutant cells under low-energy conditions (Mirouse, 2007).

Because lkb1 and ampkα mutant clones lead to very similar polarity defects and LKB1 phosphorylates AMPKα, it was asked if a constitutively active form of AMPKα could rescue the lkb1 phenotype. Therefore, transgenic lines were generated carrying a UAS-AMPKα construct, in which Threonine184 is replaced by an aspartate, which should mimic the activating phosphorylation of this site by LKB1. The expression of the AMPKα-T184D transgene in lkb1 mutant clones fully rescues their starvation-dependent polarity and overproliferation phenotypes, whereas the Gal4 driver alone has no effect. Furthermore, AMPKα-T184D-expressing mutant clones also have a normal actin cytoskeleton. Thus, the phosphomimetic version of AMPKα completely rescues the lkb1 mutant phenotype under conditions of energetic stress (Mirouse, 2007).

The recovery of null mutations in ampkα has allowed the first in vivo analysis of AMPK function in a multicellular organism, which has revealed an unexpected role for the kinase in the maintenance of epithelial polarity, but only under conditions of energetic stress. This implies that at least one of the pathways that normally maintain cell polarity cannot function when cellular energy levels are too low, and that AMPK activation compensates for this defect (Mirouse, 2007).

A surprising feature of the ampkα polarity phenotype is that it has opposite effects on the actin cytoskeleton and the cortical polarity cues. In mildly affected clones, basal actin is strongly reduced, with a corresponding increase in the amount of apical actin. In contrast, mutant clones show an expansion of the basal markers into the lateral and apical regions, as well as a loss of lateral and apical markers. Thus, the effects on actin may be independent of other polarity defects, suggesting that AMPK acts though different pathways to regulate actin and cortical polarity in opposite ways (Mirouse, 2007).

It is unclear how AMPK regulates the actin cytoskeleton, but it is possible that it acts on only one side of the cell and that the reciprocal changes on the other are caused by a change in the concentration of free G-actin or an actin nucleator, as has been shown for abl mutants during cellularization. For example, loss of AMPK could increase actin polymerization apically, thereby depleting the pool of free actin that can polymerize basally. Alternatively, ampkα mutants may prevent the formation of basal actin stress fibers, and thus increase the concentration of free actin, which enhances apical actin polymerization (Mirouse, 2007).

The cortical polarity defects of ampkα mutant clones also suggest a reciprocal relationship between the basal and apical/lateral membrane domains because the basal domain, marked by Dg, is dramatically expanded, whereas the determinants for the lateral domain (Dlg) and the apical domain (aPKC and Crb) disappear from the cortex. This suggests that there is some form of mutual antagonism between the basal and lateral domains that maintains a sharp boundary between them, as has been described for apical and lateral domains through the inhibitory phosphorylation of Baz (PAR-3) by lateral PAR-1, and of PAR-1 by apical aPKC. If this model is correct, AMPK could be required to restrict the extent of the basal domain, with the expansion of this domain in ampkα mutants leading to the exclusion of lateral and apical markers. Indeed, the overexpression of Dg has been found to cause a similar loss of apical and lateral markers to that seen in ampkα clones. Alternatively, AMPK could be necessary to maintain the localization of the apical and lateral determinants, which in turn prevent the basal domain from extending into these regions (Mirouse, 2007).

Mutations in AMPK not only disrupt the polarity of the follicle cell epithelium, but also cause the cells to overproliferate, giving rise to a tumorous phenotype. One possible explanation for this phenotype is that it is caused by the mislocalization and down-regulation of Dlg. Dlg is a member of a class of tumor suppressors in Drosophila that also includes Lgl and Scribble, and follicle cell clones mutant for any of these genes overproliferate to form invasive tumors that are similar to those formed by ampkα and lkb1 clones under low-energy conditions. Furthermore, the tumor suppressor function of these proteins is probably conserved in humans because Scribble restricts proliferation by repressing the G1/S transition, and is a target of the papilloma virus E6 oncoprotein. This may account for the observation that AMPK is required to trigger the G1/S checkpoint under conditions of energetic stress. However, it has also been shown in mammals that AMPK activates TSC2 to repress the insulin–TOR pathway (see Drosophila Tor pathway), and thus it functions as a tumor suppressor that inhibits cell growth and division (Inoki, 2003; Inoki, 2005). Loss of this repression might provide an alternative explanation for the overgrowth of ampkα mutant clones (Mirouse, 2007).

Although the molecular pathways involved remain to be elucidated, these results demonstrate that ampkα mutant cells lose their polarity under low-energy conditions and overproliferate to give rise to tumorlike growths. The activation of AMPK depends on its phosphorylation by LKB1, and loss of LKB1 produces an identical tumorous phenotype. Thus, the novel functions of AMPK reported in this work may provide a basis for the tumor suppressor function of LKB1 (Mirouse, 2007).

Energy-dependent regulation of cell structure by AMP-activated protein kinase

AMP-activated protein kinase (AMPK, also known as SNF1A) has been primarily studied as a metabolic regulator that is activated in response to energy deprivation. Although there is relatively ample information on the biochemical characteristics of AMPK, not enough data exist on the in vivo function of the kinase. Using the Drosophila model system, animals with no AMPK activity were genrated and physiological functions of the kinase investigated. Surprisingly, AMPK-null mutants are lethal with severe abnormalities in cell polarity and mitosis, similar to those of lkb1-null mutants. Constitutive activation of AMPK restores many of the phenotypes of lkb1-null mutants, suggesting that AMPK mediates the polarity- and mitosis-controlling functions of the LKB1 serine/threonine kinase. Interestingly, the regulatory site of non-muscle myosin regulatory light chain (MRLC, Spaghetti-squash; also known as MLC2 was directly phosphorylated by AMPK. Moreover, the phosphomimetic mutant of MRLC rescued the AMPK-null defects in cell polarity and mitosis, suggesting MRLC is a critical downstream target of AMPK. Furthermore, the activation of AMPK by energy deprivation was sufficient to cause dramatic changes in cell shape, inducing complete polarization and brush border formation in the human LS174T cell line, through the phosphorylation of MRLC. Taken together, these results demonstrate that AMPK has highly conserved roles across metazoan species not only in the control of metabolism, but also in the regulation of cellular structures (Lee, 2007).

The catalytic subunit of Drosophila AMPK is a single orthologue of its human and yeast counterparts, and is activated by LKB1 on energy deprivation. By imprecise excision of the EP-element (enhancer- and promoter-containing P-element) from the AMPK^G505 line, AMPK-null mutant lines, AMPK^D1 and AMPK^D2 were generated. Interestingly, all AMPK-null mutant flies are lethal before the mid-pupal stage and fail to enter adulthood, even in the presence of sufficient nutrients. Although transgenic expression of wild-type AMPK (AMPK^WT) allowed AMPK-null mutants to successfully develop into adults, the expression of kinase-dead AMPK (AMPK^KR) failed to rescue the lethality, demonstrating that the phosphotransferase activity of AMPK is crucial for its function. In summary, AMPK was found to be essential for normal development of Drosophila (Lee, 2007).

The developmental role of AMPK was further investigated by generating AMPK-null germ-line clone (AMPK-GLC) embryos, which are completely deprived of both the maternal and zygotic AMPK proteins. Surprisingly, AMPK-GLC embryos never developed into larvae, showing the requirement of AMPK during embryogenesis. In AMPK-GLC embryos, cuticle structures were severely deformed, and ventral denticle belts were missing. Furthermore, the surface of AMPK-GLC embryos was roughened and the columnar structure of the epidermis was disorganized, implicating defects in underlying epithelial structures (Lee, 2007).

To examine the embryonic epithelial structures, AMPK-GLC epithelia were examined with various polarity markers. Bazooka (Baz, apical complex marker) and β-catenin (Arm, adherens junction marker) lost their apical localization and were found in various locations around the basolateral cell surfaces. The Discs-large (Dlg, basolateral marker was also irregularly distributed throughout the epithelium in AMPK-GLC embryos. Moreover, actin staining demonstrated that the AMPK-GLC epithelium contains many unpolarized round cells that had lost contact with the underlying tissue. This disorganization of epithelial structures was not a result of cell death, because it could not be restored by overexpression of apoptosis inhibitor p35. In addition, wing discs of AMPK-null mutants also showed defective epithelial organization with ectopic actin structures in the basolateral region. These results indicate that AMPK is indispensable for epithelial integrity (Lee, 2007).

In addition, abnormally enlarged nuclei were found in some cells of AMPK-GLC embryos. Mitotic chromosome staining with anti-phospho-histone H3 (PH3) antibody demonstrated that AMPK-GLC embryos frequently contained defective mitotic cells with lagging or polyploid chromosomes. Consistently, aceto-orcein staining of squashed AMPK-null larval brains revealed polyploidy in ~30% of mitotic cells, and anti-PH3 staining showed a highly increased amount of chromosome content in some of the neuroblasts. These results indicate that AMPK is also required for the maintenance of genomic integrity (Lee, 2007).

Recently, it has been proposed that LKB1, a kinase upstream of AMPK, is involved in the regulation of epithelial polarity and mitotic cell division. Indeed, the abnormal polarity and mitosis phenotypes of lkb1-null mutants were highly similar to those of AMPK-null mutants. To test whether AMPK mediates the polarity- and mitosis-controlling functions of LKB1, constitutively active AMPK (AMPK^TD), which is catalytically active even without phosphorylation by LKB1, was expressed in lkb1-null mutants. Remarkably, AMPK^TD suppresses the epithelial polarity defects and the genomic instability of lkb1-null mutants, suggesting that AMPK is a critical downstream mediator of LKB1, controlling mitosis and cell polarity (Lee, 2007).

To understand the molecular mechanism underlying the AMPK-dependent control of mitosis and cell polarity, attempts were made to identify the downstream targets of AMPK. Intriguingly, MRLC, a critical molecule for the execution of mitosis and cell polarity establishment, contains a peptide sequence that can be phosphorylated by AMPK. Therefore, various experiments were performed to evaluate the ability of AMPK to phosphorylate MRLC. AMPK holoenzyme purified from rat liver strongly phosphorylated full-length MRLC, which was further enhanced by the addition of AMP. The phosphorylation of MRLC was more efficient than that of acetyl-CoA carboxylase 2 (ACC2), a representative substrate of AMPK, indicating that MRLC is a good in vitro substrate of AMPK. It was deduced that this phosphorylation is specifically performed by AMPK because Compound C, a specific inhibitor of AMPK, inhibited the phosphorylation, whereas ML-7, an inhibitor of another MRLC-phosphorylating kinase (MLCK), did not. A mutant form of MRLC, whose regulatory phosphorylation site (corresponding to Thr 21/Ser 22 in Drosophila and Thr 18/Ser 19 in human) was mutated into non-phosphorylatable alanines, was not phosphorylated by AMPK, suggesting that MRLC is exclusively phosphorylated at the regulatory phosphorylation site. Both the human and Drosophila forms of AMPK were able to phosphorylate MRLC from each of the respective species, which further demonstrates that the AMPK phosphorylation of MRLC is highly conserved between species (Lee, 2007).

Moreover, it was found that MRLC phosphorylation is indeed regulated by AMPK in vivo. The phosphorylation of MRLC was dramatically reduced in AMPK- and lkb1-GLC epithelia when compared with the wild-type epithelia, although the protein level of MRLC was unaffected. The reduced phosphorylation of MRLC in the AMPK-GLC epithelia was completely restored by transgenic expression of AMPK but not by overexpression of LKB1. Furthermore, in Drosophila S2 cells, energy deprivation induced by 2-deoxyglucose (2DG) enhanced MRLC phosphorylation, which was suppressed by double-strand-RNA-mediated silencing of lkb1 or AMPK. Collectively, these data strongly suggest that MRLC is specifically phosphorylated by AMPK both in vitro and in vivo (Lee, 2007).

To find out whether the phosphorylation of MRLC is critical for the physiological functions of AMPK, an active form of MRLC (MRLC^EE), whose regulatory phosphorylation site was mutated into phosphomimetic glutamates, was expressed in AMPK-GLC embryos. Strikingly, MRLC^EE rescued the epithelial polarity defects caused by the loss of AMPK, and increased the percentage of cuticle-forming embryos from ~10% to ~30%. MRLC^EE also restored the epithelial polarity defects of lkb1-null wing imaginal discs. Furthermore, the genomic polyploidy of AMPK- and lkb1-null larval brain neuroblasts was suppressed by the expression of MRLC^EE. Therefore, it is concluded that MRLC is a critical downstream target of AMPK controlling cell polarity and mitosis (Lee, 2007).

Notably, the larval brains of MRLC loss-of-function mutants (spaghetti-squash¹) showed extensive polyploidy (~40% of mitotic neuroblasts), and their imaginal discs showed severe disorganization in epithelial structure, similar to those of lkb1- and AMPK-null mutants. These phenotypic similarities further support the conclusion that MRLC is an important functional mediator of LKB1 and AMPK (Lee, 2007).

Finally, it was asked whether AMPK is critical for directing cell polarity in mammalian cells as well. To assess this, it was asked whether the activation of AMPK by 2DG treatment could induce polarization of unpolarized epithelial cells such as LS174T, which can be polarized by the activation of LKB1, in a cell-autonomous manner. Surprisingly, on 2DG treatment, LS174T cells undergo a dramatic change in cell shape to have polarized actin cytoskeleton with a brush-border-like structure. Moreover, although brush border marker villin, apical marker CD66/CEA, and basal marker CD71/transferrin were distributed throughout untreated cells, they became dramatically polarized on 2DG treatment, supporting that the activation of AMPK by energy deprivation is sufficient to induce complete polarization of LS174T cells (Lee, 2007).

It was also found that the phosphorylation of MRLC by AMPK is involved in the energy-dependent polarization of LS174T cells. Phosphorylated MRLC was colocalized with the 2DG-induced polarized actin structures, and this phosphorylation, as well as the actin polarization, was suppressed by the AMPK-specific inhibitor Compound C. Overexpression of dominant-negative AMPK (AMPK^DN) and short interfering (si)RNA-mediated inhibition of MRLC (siMRLC) also blocked the polarization, although inhibition of Par-1, another downstream kinase of LKB1, by Par-1 siRNA (siPar-1) or overexpression of dominant-negative Par-1 (Par-1^DN) failed to cause a block. More strikingly, human MRLC^EE itself was sufficient to polarize LS174T cells, even without energy deprivation, showing that phosphorylation of MRLC is critical for the AMPK-dependent polarization (Lee, 2007).

Until now, the importance of AMPK has been limited to its role as a regulator of metabolism. However, by generating the first animal model with no AMPK activity, additional functions of AMPK were characterized: AMPK regulates mitotic cell division and epithelial polarity downstream of LKB1 by controlling the activity of MRLC through direct phosphorylation. The findings revealed a link between energy status and cell structures, providing a new perspective to the diverse molecular function of AMPK. Further studies are needed on the cell-structure-controlling function of AMPK with respect to the various metabolic and physiological contexts, which may also help to understand AMPK-related diseases such as cancer and diabetes (Lee, 2007).

Drosophila alicorn, encoding the single β regulatory subunit of AMPK, is a neuronal maintenance factor protecting against activity-induced retinal degeneration

Elucidation of mechanisms that govern neuronal responses to metabolic stress is essential for the development of therapeutic strategies aimed at treatment of neuronal injury and disease. AMP-activated protein kinase (AMPK) is a key enzyme regulating cellular energy homeostasis that responds to changes in cellular energy levels by promoting energy-restorative and inhibiting energy-consumptive processes. Recent studies have suggested that AMPK might have a neuroprotective function. However, the existing evidence is contradictory and almost exclusively derived from in vitro studies based on drug treatments and metabolic stress models. To tackle these issues in vivo, the Drosophila visual system was used. A novel Drosophila mutant, alicorn (alc), is described encoding the single β regulatory subunit of AMPK. Loss of alc using the eyFlp system causes severe early-onset progressive nonapoptotic neurodegeneration in the retina, the optic lobe, and the antennae, as well as behavioral and neurophysiological defects. Retinal degeneration occurs immediately after normal neuronal differentiation, can be enhanced by exposure to light, and can be prevented by blocking photoreceptor excitation. Furthermore, AMPK is required for proper viability of differentiated photoreceptors by mechanisms unrelated to polarity events that AMPK controls in epithelial tissues. In conclusion, AMPK does not affect photoreceptor development but is crucial to maintaining integrity of mature neurons under conditions of increased activity and provides protection from excitotoxicity (Spasic, 2008).

AMP-activated protein kinase (AMPK) is an evolutionarily highly conserved key metabolic sensor of the cell. In all eukaryotic systems, it exists as a heterotrimer, composed of the catalytic α subunit and regulatory β and γ subunits, with all three being essential for the formation of an active, stable complex (Dyck, 1996; Woods, 1996). AMPK is activated through phosphorylation by at least two upstream kinases: the tumor suppressor LKB1 complex (see Drosophila Lkb1) and calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) (Hong, 2003; Woods, 2003, Woods, 2005; Hawley, 2005) but also allosterically with binding of AMP (Scott, 2004; Sanders, 2007; reviewed by Hardie, 2007a; also see Hardie's Proposed Upstream Activators and Downstream Effectors of AMPK in Neurons under Stress). Energetic stress, reflected by the rise of AMP/ATP ratio, various pathological stresses (including hypoxia, ischemia, oxidative damage, and glucose deprivation), as well as exercise and dietary hormones, activate AMPK, which responds by regulating a wide variety of cellular metabolic processes. This ultimately results in increased ATP production and decreased ATP consumption (Hardie, 2003; Spasic, 2008 and references therein).

Neurons are particularly sensitive to fluctuations in energy levels, for two reasons: (1) they are highly metabolically active cells executing a number of energy-demanding processes (e.g., maintaining ion gradients across membranes and producing action potentials), and thus they account for a high proportion of total body energy turnover, and (2) neurons have a rather inflexible metabolism in that they show poor capacity to store nutrients. High neuronal metabolic activity suggests that AMPK could play a pivotal role in neuronal maintenance. It is therefore not surprising that AMPK is highly expressed in the CNS (Turnley, 1999; Culmsee, 2001). However, the wider role of AMPK in nervous system development and function remains essentially elusive, and data available on the subject are contradictory and scarce (Hardie, 2007a; Spasic, 2008 and references therein).

Namely, there is emerging evidence that AMPK may have a neuroprotective role: AMPK is activated in the brain in response to metabolic insults such as ischemia, hypoxia, or glucose deprivation (Culmsee, 2001; Gadalla, 2004; McCullough, 2005), but it remains unclear whether the activation is beneficial. A study by Culmsee (2001) using isolated hippocampal neurons reported that AMPK activation under energy-stress conditions promotes neuronal survival (Culmsee, 2001). Additional evidence comes from Drosophila in which mutation of the AMPK γ subunit resulted in a progressive neurodegenerative phenotype (Tschape, 2002). McCullough (2005) reported that, in an in vivo CNS injury model (i.e., cerebral ischemia) as well as in an in vitro stroke model (hippocampal tissue slices subjected to oxygen-glucose deprivation), there is a global activation of AMPK. Paradoxically, pharmacological activators of AMPK had a detrimental and inhibitors a beneficial effect in these model systems (Spasic, 2008).

Understanding normal physiological functions of AMPK will provide insight into mechanisms of protection against metabolic stress and neurodegeneration. This study provides the first in vivo evidence conclusively demonstrating the involvement of AMPK in protection of mature neurons from increased metabolic activity (Spasic, 2008).

A P-element insertion line (P{lArB}K12) associated with a subtle bristle number defect was found to target CG8057 [alicorn (alc)], the single Drosophila homolog of the β subunit of AMPK (Lyman, 1996; Norga, 2003). alc has two predicted transcripts arising from two different transcription initiation sites whose existence was confirmed with Northern blot. Inverse PCR revealed that the insertion site of P{lArB}K12 was in the 5' untranslated region (UTR) of the larger alc transcript, at position -283, 591 bp away from the nearest neighboring gene, CG8788. A lethal excision δ12.125 failed to complement the deficiency covering the entire genomic region [Df(2R)Np5]. Southern blot and PCR analysis of δ12.125demonstrated a deletion of 1718 bp of the genome surrounding the P-element insertion site, including the entire first exon of both alc transcripts and 39 bp of the predicted 5' UTR of the neighboring CG8788. Quantitative real-time PCR results on the lethal excision line showed that the expression levels of CG8788 remained unaltered, whereas alc expression was entirely absent. Lack of alc expression was confirmed with whole-mount in situ hybridization of mutant embryos. Furthermore, it was found that l(2)45Ad², an ethyl methanesulfonate (EMS)-induced mutant from the same genomic region (Dockendorff, 2000), failed to complement the lethality of the excision line δ12.125. Sequencing revealed a nonsense point mutation in the second exon of the gene, replacing codon 233/112 (in transcripts RA/RB, respectively) for Arg with a premature stop signal. The resulting truncated peptide lacks the C-terminal region, which is very highly conserved between evolutionarily distant species, and contains a predicted complex-interacting region. Therefore, it is predicted that alc^Ad2 is a functional null of βAMPK (Spasic, 2008).

Both alc mutations result in lethality over a range of developmental stages, from first larval instar to pupal stage, possibly at least partially attributable to a strong maternal contribution. Lethal phase was identical in transheterozygous and hemizygous mutant combinations, further supporting the fact that alc^Ad2 and alc^δ12.125 are in fact null alleles of the same gene. Lethality of the homozygous excision allele and transheterozygous mutant forms was rescued to viable adulthood with overexpression of wild-type alc from a cDNA rescue construct, using ubiquitous daughterless- and tubulin-Gal4 driver lines. In situ hybridization of wild-type embryos using a probe generated against both alc transcripts revealed a strong maternal contribution and a broad, ubiquitous expression pattern during embryonic development (Spasic, 2008).

To circumvent organismal lethality and look at the effect of the loss of AMPK in neuronal tissue, clonal analysis of alc mutants was performed using the ey-Gal4 UAS-Flp (EGUF) system combined with a GMR-hid construct. This results in the development of an entirely mutant eye. Aside from a strong expression in larval eye-antennal imaginal discs, the eyeless enhancer is also expressed in parts of the brain, among others, the optic lobe. Although external eye morphology was grossly normal, alc EGUF-hid mutants had a striking external phenotype: although normal at eclosion, the antennae progressively accumulated an uncharacterized semitransparent organic deposit (hence, the name of the mutant, refering to the spiral horn of a unicorn), which appeared to originate from the culture media. The exact cause of this phenomenon is unclear at present, but this is likely a reflection of internal degenerative defects that are already present at day 1. Furthermore, aged mutant animals displayed behavioral changes. The antennae have been linked previously to gravitaxis in Drosophila and other insects. Indeed, when a negative geotaxis test was performed, it showed a severe impairment of the response in mutants already at day 1 and almost complete lack of response by day 7, whereas the response of control heterozygous flies was comparable with wild type. This behavioral defect could successfully be rescued by expressing the wild-type βAMPK protein (Spasic, 2008).

To investigate the consequences of the loss of AMPK in the rest of the eyeless expression domain, the morphology of the brain was examined. Paraffin brain sections of adult alc clonal mutants revealed vacuolization and neuronal degeneration in the optic lobes. This neurodegeneration had an early onset, because it was apparent already at 1 d of age. To the contrary, brains of pharate clonal mutant adults had a normal morphology. The neurodegeneration was clearly progressing, because the optic lobes of 14-d-old adults were profoundly altered. The neurodegenerative phenotype could also be rescued with wild-type alc expression (Spasic, 2008).

Next, the mechanism of neuronal death was explored. AMPK has been linked to apoptosis, paradoxically to both its activation and suppression (Ido, 2002; Russell, 2004; Shaw, 2004; Okoshi, 2008). To determine whether neuronal death in the brain is apoptotic, attempts were made to rescue using the viral apoptosis inhibitor p35. No difference was observed between p35-expressing and control mutant brains; thus, it is concluded that the observed neuronal death is independent of the p35-mediated apoptotic pathway (Spasic, 2008).

In conclusion, alc clonal mutants appeared to show a general early-onset progressive neurodegenerative phenotype in the entire expression domain of eyeless (Spasic, 2008).

Further extending these findings, the adult eye was examined for consequences of loss of alc. Initially, adult alc ey-Flp mutants were examined in a phototaxis assay in which they showed a severe impairment of phototaxis response already at 1 d of age and almost complete nonresponsiveness by day 7. As is the case with negative geotaxis, alc heterozygotes show an impairment in behavior compared with wild-type controls. This semidominance is most likely caused by haploinsufficiency. Such dosage-sensitive genetic characteristics of behavioral phenotypes have been reported previously. This behavioral defect could effectively be rescued to wild-type levels by alc expression. Subsequently, the mutant flies were subjected to ERG analysis, taking extracellular recordings that measure the response of photoreceptor neurons to a light stimulus. ERG recordings were strikingly different compared with heterozygous controls and revealed a reduction of 'on-transients' and 'off-transients,' which suggests a possible neurotransmitter release defect at the synapse between R1-R6 photoreceptors and second-order neurons in the lamina. Furthermore, decreased amplitude of depolarization for the stimulus duration indicated phototransduction defects. These ERG defects could also be reverted with a UAS-alc construct (Spasic, 2008).

To further explore the retinal morphology of adult alc mutants, semithin sections of the retina were made. Retinas homozygous for either of the alc alleles were profoundly altered, revealing extensive photoreceptor degeneration. Similar to the brain, retinal degeneration had an early onset, with elements of the phenotype present already in pharate adults, showing occasional photoreceptor (rhabdomere) loss. Interestingly, the phenotype was rapidly progressing and was significantly worse as early as day 1, with photoreceptor loss being more frequent and the appearance of vacuoles. Vacuolization of tissue and photoreceptor degeneration were further progressing with age, eventually resulting in general disorganization of ommatidial architecture at day 14. Additionally, large vesicular structures were apparent in the pigment cells. Again, the retinal phenotype was rescued by expressing wild-type βAMPK (Spasic, 2008).

In conclusion, alc clonal mutants showed phototaxis and neurophysiological defects and extensive age-dependent retinal degeneration with an early onset. Therefore, AMPK is required for viability of fully differentiated photoreceptors by mechanisms unrelated to photoreceptor development and polarity (Spasic, 2008).

The mechanism underlying neurodegeneration of mutant photoreceptors was further dissected. The sudden onset of degeneration after eclosion and its rapid progression, together with AMPK being a cellular metabolic sensor, prompted a suggestion that the degenerative phenotype is a consequence of neuronal activity. To explore this possibility, whether photoreceptor degeneration is light dependent was tested. To this end, one group of alc clonal mutants was kept in conditions of constant darkness, whereas another group was kept under controlled 12 h light/dark cycle. Notably, the phenotypes of alc dark- and light-reared EGUF-hid mutants showed striking differences: dark-reared flies at day 1 had an entirely preserved ommatidial organization. Although at 1 week of age, some signs of neurodegeneration in the dark-reared flies were obvious, the phenotype was present to a far lesser extent compared with flies kept on a light/dark cycle; vacuolization was barely present, and ommatidial architecture was grossly preserved (Spasic, 2008).

To further establish whether neuronal activity is mediating photoreceptor degeneration, whether blocking the phototransduction cascade could lead to amelioration of the phenotype was investigated. For this purpose, a mutant allele of the norpA (no receptor potential A) gene, encoding a phospholipase C necessary for phototransduction and light perception, was used. In norpA^P24 mutants, phototransduction is blocked, and the flies do not react to light and show no response to light pulse in ERG recordings. Remarkably, in a norpA^P24 mutant background, photoreceptors of alc EGUF mutants retained normal morphology, and neurodegeneration was fully suppressed at day 1. At 1 week of age, although vacuolization was present, there were no signs of photoreceptor (rhabdomere) loss and the ommatidial architecture was almost entirely preserved. Thus, the retinal neurodegenerative phenotype of alc mutants can be rescued by blocking neuronal excitation (Spasic, 2008).

Next, whether apoptosis is responsible for the observed degeneration and loss of photoreceptors was investigated. To this end, a transgenic construct was used carrying a viral apoptosis-inhibiting factor p35 to try and rescue neurodegenerative defects. With this treatment, no suppression of the phenotype was found, both vacuolization and rhabdomere loss were still apparent, and retinas were virtually indistinguishable from mutant controls lacking the p35 transgenic construct. In a separate control experiment, expression of p35 rescued the GMR-hid-induced gross morphological eye defects resulting from apoptotic cell death triggered by the hid gene. Therefore, it is concluded that the p35-mediated apoptotic pathway is not responsible for retinal defects of alc mutants, as shown previously for optic lobe degeneration (Spasic, 2008).

AMP-activated protein kinase is an evolutionarily conserved heterotrimeric enzyme whose primary role involves maintenance of energy balance in the eukaryotic cell. The structure and function of each of the subunits is conserved through evolution; however, although there are two or three genes encoding each subunit in the mammalian systems, there is but a single gene for each of them in Drosophila, making it an attractive model system to study the functions of AMPK in vivo (Pan, 2002). Although it has been the focus of much research, little is known about the precise mechanisms underlying the regulatory functions of the β and γ subunits: β acts as a scaffold via its C-terminal domain and contains a carbohydrate-binding domain that associates the mammalian enzyme complex with glycogen, whereas the gamma subunit contains the so-called Bateman domains, responsible for binding of AMP and therefore allosteric activation of AMPK (Hardie, 2007b). Finally, all three subunits are essential for the formation of an active, stable complex: a knock-out of a single subunit produces a functional knock-out of the entire enzyme (Dyck, 1996; Woods, 1996; Pan, 2002; Spasic, 2008 and references therein).

Recent research suggests that AMPK participates in physiological functions beyond those associated with responding to energy status. The fact that stimuli like dietary restriction and exercise, which have a beneficial effect on different neurodegeneration animal and cell culture models, also lead to AMPK activation (Hardie, 2003; Dasgupta, 2007) suggests possible involvement of this enzyme in neuroprotective processes. Furthermore, in the nervous system, as in most other tissues, normal aging is associated with increased amounts of oxidative stress and perturbed cellular energy metabolism involving impaired efficiency of mitochondrial ATP production. Therefore, one can imagine that the need for a functional metabolic sensor increases during aging. To date, the available information on the possible role of AMPK in neuroprotection is mostly derived from in vitro studies based on drug treatments and metabolic stress models and is contradictory (Spasic, 2008).

This study used the Drosophila visual system to address these issues and modeled changes in cellular metabolic status by influencing normal physiological activity of photoreceptors. Knock-out of AMPK using the ey-Flp system results in a rapid and severe neurodegeneration in the entire eyeless expression domain immediately on completion of normal neuronal differentiation and development. Just before eclosion, the eye-antennal system of mutant flies appears to be structurally intact, externally as well as internally, with only occasional photoreceptor loss already present. Strikingly, however, immediately after eclosion, and progressively with age, severe behavioral (phototaxis and negative geotaxis), and neurophysiological defects, optic lobe and antennal degeneration follow. It thus appears that the entire sensory system, along with parts of the brain, all deriving from the eyeless-driven AMPK knock-out succumbs to neurodegeneration with the start of neuronal activity. Although the eyeless enhancer used in the ey-Flp construct has been shown to drive expression in parts of the brain, as well as the eyes, there still is a possibility that the observed brain degeneration is (partly) a secondary, non-cell-autonomous effect. However, this remains to be proven (Spasic, 2008).

One of the striking features of AMPK clonal mutants is the extensive retinal degeneration, which can be markedly reduced by rearing flies in the dark and almost completely prevented by blocking photoreceptor excitation. Therefore, although AMPK does not seem to be required for photoreceptor development, it is crucial for maintenance and integrity of mature neurons. The fact that the mutant photoreceptors develop normally and maintain their structural integrity until they start to activate in response to light suggests that this is precisely the time when the absence of AMPK appears to have the gravest consequences. This is in agreement with the fact that activation (excitation) of neuronal cells very efficiently leads to energy depletion (Hardie, 2007a). The key factor that is responsible for bringing the energy levels back to normal in this situation is missing. Among different proposed genetic mechanisms of neurodegeneration, the AMPK phenotype illustrates how loss-of-function mutations can lead to neurodegeneration (Spasic, 2008).

In support of this paradigm, Kuramoto (2007) have reported recently that, as a result of increased neuronal activity, AMPK is activated and suppresses neuronal excitation by activating GABA_B receptors on postsynaptic neurons. Altogether, these data reveal that neurons protect themselves against excitotoxicity and that failure of such a system causes neurodegeneration. Potentially, this could be exploited to protect neurons from degeneration under adverse conditions (Spasic, 2008).

In addition, this study shows that, although AMPK regulates apico-basal cell polarity in epithelial tissues, it does not do the same in photoreceptors. Namely, mutant photoreceptors develop normally, despite their need for a precisely regulated distribution and function of cellular polarity determinants. Moreover, changes in photoreceptor polarity cannot be evoked even when subjecting the mutants to energetic stress. This is the first indication of a tissue-specific function of AMPK in this process and suggests that AMPK may not be as functionally universal in this regard as previously thought. It has been postulated before that AMPK activation in general may have cell- or tissue-specific outcomes (Ramamurthy, 2006). This could be attributable to differential expression and/or activity of its upstream regulators, as well as its downstream effectors mediating such responses. This raises intriguing questions. How is AMPK differentially regulated in cells or tissues to provide regulation of cellular polarity only in certain instances? Also, what are the mediators of the effect of AMPK on cellular polarity, and is their expression and/or activity regulated in a cell- or tissue-specific manner (Spasic, 2008)?

Finally, the exact mechanism of progressive neuronal death observed as a consequence of loss of AMPK remains unresolved. Dying by apoptosis requires a lot of energy in the form of ATP (Edinger, 2004). Therefore, apoptotic death would indeed not be expected in the situation in which cells are deprived of energy and are in addition lacking a key energy sensor. In accordance with this, both this study and Tschape (2002) in a study with the γ subunit mutant were unable to demonstrate any role of apoptosis in neurodegeneration of AMPK mutants. Conversely, autophagy (a catabolic program in which cellular constituents are degraded for energy production) is a process activated during periods of nutrient starvation and ATP depletion (Edinger, 2004). It has been shown that, under metabolic stress conditions, AMPK induces autophagy rather than apoptosis to ensure survival of cells (Liang, 2007). In addition, recent studies have indicated that autophagy is protective against neurodegeneration (Levine, 2008). In additional support of this model, Lippai (2008) has shown recently that a Drosophila P-element insertion mutant of the AMPK γ subunit results in autophagic defects during hormone-induced metamorphosis in third-instar larval fat body (Lippai, 2008). It will therefore be interesting to investigate the possible role of autophagy in neuronal loss observed in alc mutants (Spasic, 2008).

SNF4Aγ, the Drosophila AMPK γ subunit is required for regulation of developmental and stress-induced autophagy

In holometabolous insects including Drosophila a wave of autophagy triggered by 20-hydroxyecdysone is observed in the larval tissues during the third larval stage of metamorphosis. This model system was used to study the genetic regulation of autophagy. A genetic screen was performed to select P-element insertions that affect autophagy in the larval fat body. Light and electron microscopy of one of the isolated mutants (l(3)S005042) revealed the absence of autophagic vesicles in their fat body cells during the third larval stage. Formation of autophagic vesicles cannot be induced by 20-hydroxyecdysone in the tissues of mutant flies and evidence is represented demonstrating that the failure to form autophagic vesicles is due to the insertion of a P-element into the gene coding SNF4Aγ;, the Drosophila homologue of the AMPK (AMP-activated protein kinase) γ subunit. The ability to form autophagic vesicles (wild-type phenotype) can be restored by remobilization of the P-element in the mutant. Silencing of SNF4Aγ by RNAi suppresses autophagic vesicle formation in wild-type flies. An antibody was raised against SNF4Aγ. It was shown that this gene product is constitutively present in the wild-type larval tissues during postembryonal development. SNF4Aγ is nearly absent from the cells of homozygous mutants. SNF4Aγ translocates into the nuclei of fat body cells at the onset of the wandering stage concurrently with the beginning of the autophagic process. These results demonstrate that SNF4Aγ has an essential role in the regulation of autophagy in Drosophila larval fat body cells (Lippai, 2008).

p53 target genes sestrin1 and sestrin2 connect genotoxic stress and mTOR signaling

The tumor suppressor p53 is activated upon genotoxic and oxidative stress and in turn inhibits cell proliferation and growth through induction of specific target genes. Cell growth is positively regulated by mTOR, whose activity is inhibited by the TSC1:TSC2 complex. Although genotoxic stress has been suggested to inhibit mTOR via p53-mediated activation of mTOR inhibitors, the precise mechanism of this link was unknown. This study demonstrates that the products of two p53 target genes, Sestrin1 and Sestrin2 (see Drosophila Sestrin), activate the AMP-responsive protein kinase (AMPK) and target it to phosphorylate TSC2 and stimulate its GAP activity, thereby inhibiting mTOR. Correspondingly, Sestrin2-deficient mice fail to inhibit mTOR signaling upon genotoxic challenge. Sestrin1 and Sestrin2 therefore provide an important link between genotoxic stress, p53 and the mTOR signaling pathway (Budanov, 2008).

The mTOR signaling pathway is a central regulator of cell growth and survival. It is therefore not surprising that adverse environmental conditions negatively regulate cell growth by inhibiting mTOR. In addition to nutrient limitation, mTOR activity is negatively regulated by genotoxic stress and hypoxia, conditions that activate tumor suppressor p53. The ability of p53 to inhibit mTOR signaling is in line with its function as a negative regulator of cell growth and proliferation. The results described above strongly suggest that the ability of p53 to inhibit mTOR signaling depends on two of its target genes: Sesn1 and Sesn2 (Budanov, 2008).

The Sestrins belong to a small and evolutionary conserved family composed of 3 members in mammals, of which Sesn1 and 2 are stress inducible and p53 regulated. The ability of Sesn1/2 to inhibit cell growth and proliferation was attributed to their redox activity. The present work, however, demonstrates that Sesn1/2 are potent inhibitors of mTOR signaling, acting in a manner that does not depend on their redox activity, which only makes a partial contribution to their growth inhibitory activity. Sesn1 and 2 inhibit TORC1 activity towards p70S6K and 4E-BP1 in a variety of human and mouse cell lines, as well as in mouse liver. Notably, the ability of the hepatocarcinogen DEN to inhibit S6 phosphorylation is restricted to zone 3 hepatocytes, which are the main site in which it undergoes metabolic activation to become a potent alkylating agent, and this inhibitory activity is Sesn2-dependent. By inhibiting 4E-BP1 phosphorylation, Sesn2 enhances its interaction with eIF-4E and inhibits expression of growth regulatory proteins, such as cyclin D1 and c-Myc, whose translation is eIF-4E-dependent and sensitive to 4E-BP1 phosphorylation (Budanov, 2008).

The Sestrins impact TORC1 activity through the TSC1:TSC2 complex. Being a GAP for Rheb, the direct activator of TORC1, the TSC1:TSC2 complex is a central regulator of mTOR signaling. Sesn2 expression decreases Rheb GTP loading and the ability of both Sesn1 and Sesn2 to inhibit mTOR signaling is TSC2-dependent. One way to regulate TSC1:TSC2 GAP activity is through TSC2 phosphorylation, but other modes of regulation may also exist. Although the Sestrins have no effect on ERK and its target RSK or GSK3β, which can all serve as TSC2 kinases, they stimulate the activity of AMPK, a major TSC2 kinase. Furthermore, Sestrin expression enhanced TSC2 phosphorylation in live cells and this effect required the N-terminus of Sesn2, which mediates AMPKα binding. Sesn2 did not stimulate TSC1 phosphorylation and Sesn2-activated AMPK did not phosphorylate TSC1 (Budanov, 2008).

Importantly, the mTOR inhibitory activity of Sesn1/2 depends on AMPKα, whose phosphorylation at the activation loop was enhanced upon Sestrin expression. Inhibition of AMPK using compound C as well as shRNA silencing of AMPKα1 attenuated the ability of Sesn2 to inhibit mTOR signaling. Co-immunoprecipitation and gel filtration analyses revealed an interaction between Sesn2 and AMPKα, suggesting that Sestrins are engaged in formation of a large protein complex containing AMPK and TSC1:TSC2. It is proposed that Sesn1/2 induction in response to genotoxic stress results in binding of Sestrins, most likely as dimers, to AMPK and TSC1:TSC2, as well as auto-activation of AMPK through a mechanism based on induced proximity. In addition to activation of AMPK the Sestrins recruit it to phosphorylate TSC2. Phosphorylation of TSC2 correlates with enhancement of its GAP activity that leads to inhibition of Rheb and mTOR (Budanov, 2008).

Importantly, ample and clear evidence was obtained that Sesn1/2 are critical mediators of p53's ability to inhibit mTOR signaling. Using shRNA-mediated silencing it was found that both Sesn1 and Sesn2 participate in mTOR inhibition upon p53 activation in human cancer cells. Furthermore, disruption of the Sesn2 gene in mice attenuated the inhibition of p70S6K activity by the DNA-damaging agents: camptothecin in fibroblasts and DEN in hepatocytes. In both cases inhibition of p70S6K was p53-mediated, but unlike the p53 deficiency, the absence of Sesn2 has no effect on induction of p21^Waf1, another p53 target gene. Thus, Sesn2 (and presumably Sesn1) seems to mediate only one aspect of p53 signaling -- inhibition of mTOR. Correspondingly, the growth-inhibitory activity of Sesn2 is not as strong as that of p53, which has additional targets with anti-proliferative activity, such as p21^Waf1 (Budanov, 2008).

p53 deficiency and activation of mTOR signaling are hallmarks of human cancer. Several mechanisms account for mTOR activation in cancer, including activation of Ras, PI3K and AKT and inactivation of tumor suppressors that negatively regulate these molecules: PTEN, TSC1, TSC2 and LKB1. Although p53 can induce expression of several negative regulators of mTOR, including PTEN, TSC2, AMPKβ1 and IGF-BP3 in a cell type-dependent manner, the results demonstrate that p53-mediated inhibition of mTOR depends mainly on Sesn1 and 2 in mouse fibroblasts and certain human cancer cell lines and on Sesn2 in mouse liver (Budanov, 2008).

Inhibition of mTOR suppresses cell growth and proliferation. Sesn2 was known to inhibit cell proliferation, but its mechanism of action was heretofore unknown. The results strongly suggest that Sesn1 and Sesn2 exert their growth inhibitory effect via mTOR and may cooperate with other anti-proliferative p53 targets, such as p21^Waf1. Interestingly, the SESN1 (6q21) and SESN2 (1p35) loci are frequently deleted in a variety of human cancers, suggesting they harbor one or more tumor-suppressors. Sesn2 deficiency was found to render murine fibroblasts more susceptible to oncogenic transformation and this effect may depend on mTOR inhibition. Hence, SESN1 and SESN2 may indeed be important components of the tumor suppressor network activated by p53 (Budanov, 2008).

In summary, while more remains to be learned about Sestrin biology and mechanism of action, the results establish these proteins as critical links between p53 and mTOR that enable p53 to inhibit cell growth (Budanov, 2008).

Sestrin as a feedback inhibitor of TOR that prevents age-related pathologies

Sestrins are conserved proteins that accumulate in cells exposed to stress, potentiate adenosine monophosphate-activated protein kinase (AMPK), and inhibit activation of Target of rapamycin (TOR). The abundance of Drosophila sestrin (dSesn) is increased upon chronic TOR activation through accumulation of reactive oxygen species that cause activation of c-Jun amino-terminal kinase and transcription factor Forkhead box O (FoxO). Loss of dSesn resulted in age-associated pathologies including triglyceride accumulation, mitochondrial dysfunction, muscle degeneration, and cardiac malfunction, which were prevented by pharmacological activation of AMPK or inhibition of TOR. Hence, dSesn appears to be a negative feedback regulator of TOR that integrates metabolic and stress inputs and prevents pathologies caused by chronic TOR activation that may result from diminished autophagic clearance of damaged mitochondria, protein aggregates, or lipids (Lee, 2010).

Target of rapamycin (TOR) is a key protein kinase that regulates cell growth and metabolism to maintain cellular and organismal homeostasis. Insulin and insulin-like growth factors are major TOR activators that operate through phosphoinositide 3-kinase (PI3K) and the protein kinase AKT. Conversely, adenosine monophosphate-activated protein kinase (AMPK), which is activated upon energy depletion, caloric restriction (CR), or genotoxic damage, is a stress-responsive inhibitor of TOR activation. TOR stimulates cell growth and anabolism by increasing protein and lipid synthesis through p70 S6 kinase (S6K), eukaryotic translation initiation factor 4E-binding protein (4E-BP), and sterol response element binding protein (SREBP) and by decreasing autophagic catabolism through phosphorylation-mediated inhibition of ATG1 protein kinase. Persistent TOR activation is associated with diverse pathologies such as cancer, diminished cardiac performance, and obesity-associated metabolic diseases. Conversely, inhibition of TOR prolongs life span and increases quality of life by reducing the incidence of age-related pathologies. The antiaging effects of CR could be due to inhibition of TOR (Lee, 2010 and references therein).

Sestrins (Sesns) are highly conserved proteins that accumulate in cells exposed to stress, lack obvious domain signatures, and have poorly defined physiological functions. Mammals express three Sesns, whereas Drosophila melanogaster and Caenorhabditis elegans have single orthologs. In vitro, Sesns exhibit oxidoreductase activity and may function as antioxidants. Independently of their redox activity, Sesns lead to AMPK-dependent inhibition of TOR signaling and link genotoxic stress to TOR regulation (Badanov, 2008). However, Sesns are also widely expressed in the absence of exogenous stress, and in Drosophila, expression of Drosophila sestrin (dSesn) is increased upon maturation and aging. Given the redundancy between mammalian Sesns, the importance of Sesns as regulators of TOR function was tested in Drosophila. Both gain- and loss-of-function dSesn mutants were created. Analysis of these mutants revealed that dSesn is an important negative feedback regulator of TOR whose loss results in various TOR-dependent, age-related pathologies (Lee, 2010).

Persistent TOR activation in wing discs by a constitutively active form of the insulin receptor (InR^CA) resulted in prominent dSesn protein accumulation, which is not seen in a dSesn-null larvae. InR^CA also induced accumulation of dSesn RNA, indicating that dSesn accumulation is due to increased transcription or mRNA stabilization. Since dSesn accumulation was restricted to cells in which TOR was activated, the response is likely to be cell autonomous. dSesn was also induced when TOR was chronically activated by overexpression of the small guanine triphosphatase Rheb, clonal loss of phosphatase and tensin homolog (PTEN), or tuberous sclerosis complex 1 (TSC1). Dominant-negative PI3K (PI3K^DN) or TOR (TOR^DN) inhibited dSesn accumulation caused by overexpression of InR^CA, but inactive ribosomal S6 protein kinase (S6K, S6K^DN) and hyperactive 4E-BP (4E-BP^CA), two downstream TOR effectors, did not. Furthermore, dorsal-specific expression of activated S6K^CA or loss of 4E-BP activity failed to induce dSesn expression, indicating that TOR regulates expression of dSesn through different effector(s) (Lee, 2010).

In mammals, transcription of Sesn genes is increased in cells exposed to oxidative stress, and reactive oxygen species (ROS) accumulation, detected by oxidation of dihydroethidium (DHE), was observed in the same region of the imaginal discs in which InR^CA or Rheb were expressed. InR^CA-induced accumulation of ROS was blocked by coexpression of either PI3K^DN or TOR^DN, but not S6K^DN or 4E-BP^CA, revealing TOR’s role in ROS accumulation. Wing-disc clones in which TOR was activated by loss of TSC1 also exhibited ROS accumulation, confirming that TOR-dependent ROS accumulation is cell-autonomous. Expression of the ROS scavengers catalase or peroxiredoxin inhibited InR^CA-induced accumulation of dSesn. Feeding animals with vitamin E, an antioxidant, also prevented dSesn induction caused by TSC1 loss (Lee, 2010).

Forkhead box O (FoxO) and p53 are ROS-activated transcription factors that control mammalian Sesn genes. The dSesn locus contains eight perfect FoxO-response elements, a frequency 25 times higher than that expected on the basis of random distribution. Overexpressed FoxO or p53 could both increase expression of the dSesn gene. However, InR^CA caused accumulation of dSesn in a p53-null background, but not in a FoxO-null background, indicating that TOR-activated FoxO is likely to be the regulator of dSesn gene transcription. Accumulation of dSesn in response to Rheb overexpression was also FoxO-dependent (Lee, 2010).

In dorsal wing disc cells, where ROS accumulated in response to InR^CA, c-Jun N-terminal kinase (JNK), a protein kinase that phosphorylates FoxO, was also activated. JNK activation was diminished in cells overexpressing catalase, suggesting that it depends on TOR-induced accumulation of ROS. Mitogen-activated protein kinase kinase 7-mediated activation of JNK also resulted in accumulation of dSesn, as did overexpression of mammalian STE20-like kinase 1 (MST1), another protein kinase that phosphorylates FoxO. However, only JNK^DN (but not Mst1^DN) inhibited InR^CA-mediated accumulation of dSesn. Collectively, these data suggest that dSesn transcription is increased upon chronic TOR activation through ROS-dependent activation of JNK and FoxO (Lee, 2010).

To determine effects of dSesn on cell growth, a major function of TOR, dSesn was overexpressed in dorsal wings. This resulted in a dose-dependent phenotype in which the wing bends upward, indicating suppressed dorsal tissue growth. A dSesn^C86S variant, in which the cysteine required for oxidoreductase activity was mutated (C86S, Cys⁸⁶->Ser⁸⁶), still conferred this phenotype when expressed in amounts similar to those of wild-type dSesn (dSesn^WT). Cell number and size were measured in a dorsal wing region defined by the L3, L4, C1, and C2 veins. Although the size of this area was significantly reduced by dSesn expression, the cell number remained unchanged, showing that decreased cell size can account for dSesn suppression of tissue growth. Overexpression of dSesn also reduced cell size in larval wing discs and adult eyes. Thus, dSesn inhibits cell growth without affecting cell proliferation and does so independently of its redox activity (Lee, 2010).

When dSesn was expressed with InR^CA or Rheb, it suppressed the hyperplastic phenotypes caused by these TOR activators. Both eye and individual ommatidia sizes were significantly reduced. dSesn also inhibited InR^CA- or Rheb-induced phosphorylation of TOR targets S6K and 4E-BP. In mammalian cells, dSesn enhanced AMPK-induced phosphorylation of TSC2 and inhibited S6K activity through TSC2, just as mSesn2 does (Budanov, 2008). In Drosophila wings, dSesn-induced growth suppression was attenuated by reduced gene dosage of TSC1, TSC2, or AMPK, although reduced dosage of these genes alone did not affect normal growth. Expression of mSesn1/2 in flies also reduced normal and InR^CA-induced hyperplastic growth (Lee, 2010).

Expression of InR, constitutively active PI3K (PI3K^CA), AKT, or S6K^CA in dorsal cells of the wing caused an overgrowth phenotype in which the wing bends downward. dSesn expression reversed this effect of overexpressed InR, PI3K^CA, and AKT, but not that of S6K^CA, suggesting that dSesn inhibits TOR downstream of AKT. Conversely, dorsal wing-specific expression of PTEN and InR^DN, PI3K^DN, or S6K^DN caused wings to bend upward, and this effect was enhanced by dSesn (Lee, 2010).

Although dSesn-null flies did not exhibit developmental abnormalities, the growth-promoting effect of overexpressed InR or AKT was enhanced in dSesn-null background, suggesting that endogenous dSesn restricts TOR activation and its growth-promoting effect. Loss of dSesn, however, did not enhance S6K-stimulated cell growth or decrease growth suppression by overexpressed InR^DN or S6K^DN. These findings indicate that Sesn is an evolutionarily conserved inhibitor of TOR signaling that acts via the AMPK-TSC2 axis (Lee, 2010).

Fat bodies from dSesn-null third-instar larvae contained more lipids than did those of WT animals. dSesn-null adults also contained more triglycerides, which were decreased after ectopic expression of dSesn^WT or dSesn^CS. Thus, the TOR-inhibitory function of dSesn, rather than its antioxidant activity, appears to affect metabolic control. Congruently, dSesn-null fat bodies showed decreased AMPK and increased TOR activities. Pharmacological manipulation strengthened this conclusion; feeding dSesn-null mutants with AMPK-activators such as 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) or metformin, or the TOR-inhibitor rapamycin reduced triglyceride accumulation (Lee, 2010).

Expression of the gene-encoding transcription factor dSREBP and its targets, which encode fatty acyl coenzyme A (CoA) synthetase, fatty acid synthase, acetyl CoA carboxylase, and acetyl CoA synthetase, was significantly increased (20 to 70%) in dSesn-null mutants. However, the peroxisome proliferator-activated receptor γ coactivator 1 (dPGC-1) gene and some lipolytic genes showed decreased expression. This is consistent with reports that dSREBP and dPGC-1 (spargel; CG9809)are inversely regulated by TOR and AMPK to properly control lipid metabolism (Lee, 2010).

Age-related decline in heart performance is another phenotype associated with TOR hyperactivity in insects and mammals. In WT flies, the heart beats in a highly regular manner, but in dSesn-null mutants, heart function was compromised, as manifested by arrhythmia and decreased heart rate. Slowing of heart rate reflected expansion of the diastolic period, as observed in aged or TOR-activated flies. These defects were largely prevented by feeding flies AICAR or rapamycin, indicating that they are caused by low activity of AMPK or high TOR activity. Vitamin E feeding or catalase expression suppressed the arrhythmia caused by loss of dSesn, but not the decrease in heart rate, suggesting that TOR-induced oxidative stress contributes to the arrhythmic phenotype. Analysis of F-actin revealed structural disorganization of myofibrils in dSesn-null hearts, suggesting that cardiac muscle degeneration may cause some of the functional defects in dSesn-null hearts. Reflecting this structural abnormality, dSesn-null hearts were dilated during both the diastolic and systolic phases, and this was prevented by AICAR or rapamycin (Lee, 2010).

Heart-specific depletion of dSesn caused cardiac malfunction similar to that seen in dSesn-null mutants. Heart-specific depletion of AMPK also caused cardiac malfunction, but this was not alleviated by AICAR administration, supporting the notion that dSesn maintains normal heart physiology through AMPK activation (Lee, 2010).

dSesn mRNA and protein are abundant in the adult thorax, which is mostly composed of Mesoderm. mSesn1 is also highly expressed in skeletal muscle (Velasco-Miguel, 1999). Therefore, whether dSesn has a role in maintaining muscle homeostasis was tested. 20-day-old dSesn-null flies showed degeneration of thoracic muscles with loss of sarcomeric structure, including discontinued Z discs, disappearance of M bands, scrambled actomyosin arrays, and diffused sarcomere boundaries. Such defects are only partially observed in very old WT flies (~90 days) and were not found in young (5-day-old) dSesn-null muscles. Thus, the dSesn-null skeletal muscle appears to undergo accelerated age-related degeneration (Lee, 2010).

Despite its normal appearance, muscle from 5-day-old dSesn-null flies exhibited mitochondrial abnormalities, including a rounded shape, occasional enlargement, and disorganization of cristae, which were also observed in 20-day-old mutants. Mitochondrial dysfunction can result in excessive generation of ROS leading to other abnormalities. dSesn-null muscles exhibited increased accumulation of ROS, revealed by more intense DHE fluorescence and reduced cis-aconitase activity, which was associated with muscle cell death. Furthermore, the muscle defects were prevented by vitamin E feeding, underscoring the role of ROS in muscle degeneration (Lee, 2010).

Expression of exogenous dSesn^CS, devoid of redox activity, prevented muscle degeneration, suggesting again that regulation of AMPK-TOR by dSesn, rather than intrinsic redox activity, is of importance. Feeding animals with AMPK activators prevented muscle degeneration in dSesn-null mutants, and depletion of AMPK in skeletal muscles caused severe degeneration of mitochondrial and sarcomeric structures. Treatment of animals with rapamycin also prevented muscle degeneration in dSesn-null flies. Thus, dSesn-dependent control of AMPK-TOR signaling is essential for prevention of mitochondrial dysfunction and maintenance of muscle homeostasis during aging (Lee, 2010).

It was noticed that dSesn-null muscles accumulated polyubiquitin aggregates, which are hallmarks of defective autophagy. To test whether decreased autophagy brought about by excessive and prolonged TOR activity might cause muscle degeneration, expression was silenced of ATG1, an essential component of the autophagic machinery, which is inhibited by TOR. This caused a decline in cardiac performance, as well as degeneration and mitochondrial abnormalities in skeletal muscle. These results suggest that TOR up-regulation caused by dSesn loss inhibits autophagy needed to eliminate ROS-producing dysfunctional mitochondria, which may contribute to muscle degeneration. Consistent with this view, ATG1 silencing resulted in ROS accumulation in wing discs (Lee, 2010).

These results identify Sesn as a negative feedback regulator of TOR function. In mammalian cells, increased expression of mSesns in response to genotoxic stress leads to inhibition of TOR activity through activation of AMPK (Budanov, 2008). This study now shows that transcription of the dSesn gene is increased upon chronic TOR activation through JNK and FoxO in a manner dependent on ROS accumulation. Although transient InR activation inhibits FoxO through its phosphorylation by AKT, this study finds that chronic TOR activation overcomes this inhibition and results in nuclear translocation of FoxO, which increases dSesn transcription. In turn, dSesn suppresses metabolic dysfunction and age-related tissue degeneration brought about by hyperactivated TOR. Although dSesn can inhibit TOR-stimulated cell growth, this analysis points to its most important function being the maintenance of metabolic homeostasis and prevention of TOR-induced tissue degeneration. The three major functions of dSesn revealed by this study -- suppression of lipid accumulation, prevention of cardiac malfunction, and protection of muscle from age-related degeneration -- are adversely affected by obesity, lack of exercise, and aging, which make a disproportional contribution to health problems in developed and rapidly developing societies (Lee, 2010).

Whereas TOR controls cell growth mostly through inhibition of 4E-BP and activation of S6K kinase, its ability to induce dSesn expression depends on ROS accumulation, which the results suggest is a pathophysiological aberration caused by TOR hyperactivation that is normally antagonized by dSesn. However, the previously described redox function of Sesn is not required for its protective role. TOR-induced accumulation of ROS has been observed in yeast and hematopoietic cells, but the molecular mechanism underlying this phenomenon and its physiological and pathophysiological importance were unknown. The current results suggest that TOR-stimulated production of ROS, which is needed for accumulation of dSesn, is independent of two of the major TOR targets (4E-BP and S6K) and instead may result from TOR-mediated inhibition of physiological autophagy, a process that eliminates ROS-producing dysfunctional mitochondria. Nonetheless, inhibition of 4E-BP also contributes to the pro-aging effects of TOR by suppressing translation of several mitochondrial proteins and by accelerating age-related cardiac malfunction at young ages, which is reminiscent of the observed cardiac defects seen in dSesn-null flies. Although TOR activates SREBP, which may contribute to lipid accumulation in dSesn-null flies, autophagy promotes lipid elimination. Thus, decreased autophagy may also contribute to triglyceride accumulation. Hence, the different degenerative phenotypes exhibited by dSesn-null flies are due to the cumulative effects of several biochemical and cell biological defects caused by hyperactive TOR, including reduced autophagy and reduced function of 4E-BP. Both basal physiologic autophagy and 4E-BP function are enhanced by calorie restriction, which prevents aging-related pathologies. In the future, it will be of interest to determine the contribution of Sesn to these antiaging effects (Lee, 2010).

Mitochondrial regulation of cell cycle progression during development as revealed by the tenured mutation in Drosophila

The precise control of the cell cycle requires regulation by many intrinsic and extrinsic factors. Whether the metabolic status of the cell exerts a direct control over cell cycle checkpoints is not well understood. A mutation was isolated in tenured (tend), encoding cytochrome oxidase subunit Va. This mutation causes a drop in intracellular ATP to levels sufficient to maintain cell survival, growth, and differentiation, but not to enable progression through the cell cycle. Analysis of this gene in vivo and in cell lines shows that a specific pathway involving AMPK and p53 is activated that causes elimination of Cyclin E, resulting in cell cycle arrest. In multiple tissues the mitochondrion has a direct and specific role in enforcing a G1-S cell cycle checkpoint during periods of energy deprivation (Mandal, 2005).

This study describes a mitotic checkpoint that is activated in periods of lowered mitochondrial function. A null mutation in CoVa, a nuclear-encoded component of the mitochondrial electron transport chain, lowers intracellular ATP to about 40% of normal. This ATP level is sufficient to support cell survival, growth, and differentiation, and it allows genetic dissection of the pathway connecting mitochondrial function to cell division. The 60% reduction in ATP levels in mutant cells activates the energy sensor AMPK, presumably due to an increase in cellular AMP levels. In turn, AMPK activates the cell cycle checkpoint regulator p53 and brings about a cell cycle arrest by reducing levels of the rate-limiting protein Cyclin E. Although it might be assumed that mutations in mitochondrial proteins would cause a general slowdown of all cellular processes, the results show that tend mutant cells are remarkably healthy and are capable of differentiation and morphogenesis. In the eye disc, tend mutant cells adopt their appropriate fate, transcribe genes related to proliferation and differentiation control, and are capable of extensive morphological changes, such as projecting axons to their target regions in the brain. The only cellular dysfunction that was detected upon the 60% drop in ATP levels in tend mutants is the block in cell cycle. Furthermore, no increase was observed in apoptosis in tend mutant cells, although it is likely that other mitochondrial mutations that cause a more precipitous drop in ATP levels would activate cell death mechanisms. It is concluded that a mechanism involving Cyclin E operates to block the cell cycle specifically at the G1-S transition point in response to a limiting threshold in its ATP level (Mandal, 2005).

Cell division is an energy-intensive process, and inhibition of the cell cycle by an ATP-dependent checkpoint is critical. Checkpoints are surveillance mechanisms that respond to stress and activate processes that maintain viability. AMPK and p53 are not required for regulating cell cycle progression under normal growth conditions in wild-type flies. As a result, loss-of-function mutations in either AMPKγ or p53 have a wild-type eye phenotype. However, AMPK and p53 do function to control the cell cycle in response to low ATP by triggering downregulation of Cyclin E protein. As has been described for other checkpoints, it has been possible to override this cell cycle arrest. This was achieved by making double mutant clones of tend and p53 or tend and AMPKγ. The result is a dramatic rescue of the BrdU incorporation phenotype as well as Cyclin E expression in the double mutant cells. Previous work has shown Drosophila p53 to have a role in irradiation-mediated cell death. In contrast, mammalian p53 has been shown to have a function in both cell cycle and apoptosis. Drosophila p53 also functions in cell cycle arrest under conditions of modest loss of metabolic function. It is likely that the triggering of apoptotic pathways by irradiation requires a higher threshold of p53 function. This notion is consistent with work in mammalian systems in which small changes in p53 levels or activity trigger a cell cycle arrest. Moreover, as the levels increase the cells turn on the apoptotic machinery (Mandal, 2005).

In addition to CoVa, mutations in several other mitochondrial proteins, primarily large and small subunits of ribosomal proteins (mRpLs and mRpSs), also show a BrdU incorporation block similar to that seen in tend. While the details of the molecular pathways affected in these other mutants remain to be investigated, it is likely that these mutations affect the translation of proteins encoded by the mitochondrial genome and as a result prevent the electron transport chain from functioning optimally. Since multiple genes encode proteins of the ribosomal subunits, their functions may be redundant, and a mutation in any one component may only partially reduce mitochondrial translation. However, not all mRp mutations show cell cycle arrest. It is speculated that when mutations are made in each mitochondrial protein and in combinations, it will be possible to identify mechanisms that create a balance between cell proliferation, cell growth, and cell death. In vivo observations of deprivation of mitochondrial function are similar to the observations made in cell culture experiments in which mouse embryonic fibroblasts are exposed to different levels of glucose. Whereas cells completely deprived of glucose die, cells receiving 0.5 mM glucose show a p53-dependent cell cycle arrest similar to that seen upon partial ATP deprivation in the current studies (Mandal, 2005).

The downregulation of Cyclin E in response to mitochondrial dysfunction is posttranscriptional. It is not yet clear whether this is due to reduced translation of the Cyclin E transcript or accelerated degradation of the Cyclin E protein. In S. cerevisiae, Cln3, a Cyclin critical for the G1-S transition, is regulated at the translational level in response to the absence of a fermentable carbon source. A similar mechanism could potentially provide a means of altering Cyclin E protein levels without affecting other protein products in the cell. In contrast, several Ubiquitin ligases, including Archipelago, Sina, and Ebi, have been associated with normal Cyclin E degradation during the cell cycle. However, in preliminary studies of the kind that allowed identification of AMPK and p53 as downstream components of tend, no role has been detected for archipelago, sina, or ebi in this process. This is not surprising since the Drosophila genome has over 50 potential genes encoding members of the E3 Ubiquitin ligase complex. Finally, in mammalian systems, p53-activated G1-S arrest involves transcriptional activation of the CDK inhibitor p21. The only known homolog of the p27/p21 family of proteins in Drosophila is Dacapo, which is not upregulated in tend mutant cells. Although, no other p21-like gene has been identified, it remains a possibility that a protein with low homology to p21 is involved as a CDK inhibitor. Given the tools available in Drosophila, future modifier screens should reveal the identity of the missing component between p53 and Cyclin E (Mandal, 2005).

An interesting finding from this work is that tend cells are not compromised in their size. In fact, mutant cells show a slight (13%) increase in size compared to their wild-type counterparts. Previous work has shown that AMPK activation inhibits TOR function, and it was therefore conceivable that the cell cycle arrest phenotype in tend could be a consequence of a similar growth arrest. Instead, this study shows that tend mutants have a phenotype complementary to that of TOR. Cells mutant for TOR are smaller in size and show normal BrdU incorporation, while tend cells are slightly larger than wild-type and are affected in the G1-S transition step of cell division (Mandal, 2005).

This study is an in vivo demonstration of a mitochondrial regulation of a checkpoint blocking cell cycle progression. Activation of this regulatory mechanism to block the cell cycle will allow a cell to weather a period of energy deprivation by pausing in the cell cycle and resuming proliferation upon the return of energy sources. Periods of energy insufficiency can be due to a variety of reasons, including limited oxygen availability. Hypoxia diminishes the cell's oxidative phosphorylation capacity and can cause cell cycle arrest in a number of species, including Drosophila. Similarly, it has been suggested that the ability of cells deep inside solid tumors to withstand radiation as well as chemotherapy is due to hypoxia-induced G1 arrest. It is tempting to speculate that cells could use this mode of cell cycle arrest to their advantage during normal development. Hematopoetic stem cells in the bone marrow are distributed along a gradient of oxygen; stem cells residing in more hypoxic conditions cycle slowly, while proliferating progenitor cells are in normoxic regions. It will be interesting to determine in future studies if the cell cycle arrest observed in stem cells utilizes pathways that described in this study for CoVa (Mandal, 2005).

The neurodegeneration mutant löchrig interferes with cholesterol homeostasis and Appl processing

The novel Drosophila mutant löchrig (loe: SNF4/AMP-activated protein kinase gamma subunit) shows progressive neurodegeneration and neuronal cell death, in addition to a low level of cholesterol ester. loe affects a specific isoform of the gamma-subunit of AMP-activated protein kinase (AMPK), a negative regulator of hydroxymethylglutaryl (HMG)-CoA reductase and cholesterol synthesis in vertebrates. Although Drosophila cannot synthesize cholesterol de novo, the regulatory role of fly AMPK on HMG-CoA reductase is conserved. The loe phenotype is modified by the level of HMG-CoA reductase and suppressed by the statin-induced inhibition of this enzyme; statin has been used for the treatment of Alzheimer patients. In addition, the degenerative phenotype of loe is enhanced by a mutation in amyloid precursor protein-like (APPL), the fly homolog of the human amyloid precursor protein involved in Alzheimer's disease. Western analysis has revealed that the loe mutation reduces APPL processing, whereas overexpression of Loe increases it. These results describe a novel function of AMPK in neurodegeneration and APPL/APP processing that could be mediated through HMG-CoA reductase and cholesterol ester (Tschäpe, 2002).

For a long time cholesterol metabolism has been investigated in peripheral cells, yet relatively little is known about it in brain cells. This is all the more surprising as the brain is the organ richest in cholesterol. Most cells in the body take up the required amount of cholesterol via the LDL or VLDL (low- and very low-density lipoprotein) receptor pathway. After uptake, the lipoproteins are degraded and the cholesterol released within the cell where it can be either used as free cholesterol or stored in the form of cholesterol ester. This transport mechanism is highly conserved in vertebrates and invertebrates. In addition, vertebrate cells can produce cholesterol by de novo synthesis in the endoplasmic reticulum. Due to the blood-brain barrier, brain cells are unable to receive their supply of lipoproteins from the plasma and it has been suggested that only very little is supplied by uptake. At least oligodendrocytes seem to meet their demand for cholesterol by de novo synthesis. Nevertheless, the cerebrospinal fluid contains special lipoproteins, the apolipoproteins apoE and apoAI, and most probably these brain lipoproteins are not involved in the transport of cholesterol to and from the brain but rather in the redistribution of cholesterol within the brain (Tschäpe, 2002 and references therein).

Cholesterol regulates the physical properties of the cell membrane, and its level is therefore tightly controlled. Recent work has shown that cholesterol plays a role in membrane compartmentalization and in the formation of lipid rafts. This important function might be the reason for the connection between cholesterol and neurodegeneration. Studies have shown that the cholesterol level influences the production of the pathogenic Aß peptide, which is produced from the amyloid precursor protein (APP) by cleavage through ß- and gamma-secretase. It has been suggested that Aß processing occurs within rafts, whereas the non-amyloidogenic alpha-processing occurs outside. Cholesterol synthesis in neurons is regulated by hydroxymethylglutaryl-CoA (HMG-CoA) reductase, which again has been connected to Alzheimer's disease. Inhibition of this enzyme by statins not only reduces cholesterol synthesis but also inhibits ß-secretase cleavage of APP. In addition, clinical studies indicate that patients treated with statins have a decreased prevalence of Alzheimer's disease. HMG-CoA reductase activity is negatively regulated via phosphorylation through the AMP-activated protein kinase (AMPK), a heterotrimeric complex, consisting of the catalytic alpha-subunit and ß- and gamma-subunits, found in all eukaryotes (Tschäpe, 2002).

The Drosophila mutant löchrig (loe) disrupts a specific isoform of the AMPK gamma-subunit, which leads to a low level of cholesterol ester together with a strong neurodegenerative phenotype. loe interacts genetically with HMG-CoA reductase and influences processing of the ß-amyloid protein precursor-like (APPL) gene. Although the regulation and most downstream targets of HMG-CoA reductase are conserved, this enzyme is not involved in cholesterol synthesis in insects, because they cannot synthesize cholesterol de novo. The loe mutant now shows that HMG-CoA reductase and its regulator AMPK are also involved in neurodegeneration in insects. The low level of cholesterol ester suggests that the mediator could be cholesterol ester rather than cholesterol, which might be important in the context of Alzheimer's disease because the level of cholesterol ester has been directly correlated with Aß production in cell culture experiments (Tschäpe, 2002).

loe was isolated from a collection of P-element insertion lines. About 800 lines that have a shortened adult life span were aged and screened histologically for signs of neurodegeneration. Two of these lines showed severe vacuolization of the central nervous system (CNS) which increased with aging, and one of them was named löchrig (the German term for 'full of holes'). The vacuolar pathology is most prominent around the central complex and in the central parts of the brain, while the optic lobes are less affected. Developmental studies have suggested that the vacuolization and degeneration in loe are confined to differentiated, probably synaptically active neurons, whereas neuroblasts and developing neurons are unaffected (Tschäpe, 2002).

cDNAs of the loe gene represent at least six alternatively spliced transcripts for the Drosophila gamma-subunit of AMPK. The different mRNAs encode at least three different protein isoforms, all sharing the same C-terminus while varying in their N-terminal part. The C-terminus includes the so-called CBS (cystathionine-ß-synthase) domains that are highly conserved between yeast, mammals and Drosophila. Interestingly, a region in the unique N-terminus of the LoeI isoform shows homology to the X11alpha protein which can bind to the APP protein (Borg, 1998); LoeI and X11 are 28% identical and 41% similar over a stretch of 80 amino acids. The P-element is inserted in the seventh intron of this transcript and 38 bp upstream of the transcription start site of LoeII, suggesting that one or two transcripts are affected by the insertion (all other transcripts are >10 kb downstream of the insertion site and therefore most probably are not affected by the P-element). A small deletion of 1.3 kb was created around the insertion site, removing exon 1 of the LoeII transcript, and these flies do not show a degeneration phenotype. This indicates that LoeII is not required for CNS integrity (Tschäpe, 2002).

To assess whether the loe mutation influences cholesterol metabolism, a role described for AMPK, the lipid composition of fly heads was measured. The analysis of phospholipids, triglycerides and free cholesterol did not reveal any significant differences between 1- to 5-day-old wild-type and mutant flies. The amount of cholesterol ester, however, was reduced by ~40%. Expressing LoeI in neurons restored the wild-type level of cholesterol ester in the mutant, confirming the role of Loe/AMPK in cholesterol homeostasis. The expression of LoeI restores the cholesterol ester level as well as the neurodegenerative phenotype, directly connecting cholesterol ester and neurodegeneration in the loe mutant. These results reveal an involvement of AMPK in cholesterol ester levels in the brain independent of de novo cholesterol synthesis. In peripheral tissues, vertebrate AMPK inhibits the activation of a hormone-sensitive lipase, an enzyme involved in the breakdown of cholesterol ester. A conserved regulatory pathway in the brain could account for the decreased amount of cholesterol ester (Tschäpe, 2002 and references therein).

A homologue of AMP-activated protein kinase in Drosophila melanogaster is sensitive to AMP and is activated by ATP depletion

Single genes have been identified encoding homologues of the α, β and γ subunits of mammalian AMP-activated protein kinase (AMPK) in the genome of Drosophila. Kinase activity could be detected in extracts of a Drosophila cell line using the SAMS peptide, which is a relatively specific substrate for the AMPK/SNF1 kinases in mammals and yeast. Expression of double stranded (ds) RNAs targeted at any of the putative α, β or γ subunits ablated this activity, and abolished expression of the α subunit. The Drosophila kinase (DmAMPK) was activated by AMP in cell-free assays (albeit to a smaller extent than mammalian AMPK), and by stresses that deplete ATP (oligomycin and hypoxia), as well as by carbohydrate deprivation, in intact cells. Using a phosphospecific antibody, it was shown that activation was associated with phosphorylation of a threonine residue (Thr-184) within the 'activation loop' of the α subunit. A homologue of acetyl-CoA carboxylase (DmACC) was identified in Drosophila and, using a phosphospecific antibody, it was shown that the site corresponding to the regulatory AMPK site on the mammalian enzyme became phosphorylated in response to oligomycin or hypoxia. By immunofluorescence microscopy of oligomycin-treated Dmel2 cells using the phosphospecific antibody, the phosphorylated DmAMPK α subunit was mainly detected in the nucleus. These results show that the AMPK system is highly conserved between insects and mammals. Drosophila cells now represent an attractive system to study this pathway, because of the small, well-defined genome and the ability to ablate expression of specific gene products using interfering dsRNAs (Pan, 2002).

Although there have been previous studies of the homologous SNF1 system in yeast and the SNF1-related protein kinases in higher plants, this is the first study of AMPK in the animal kingdom outside of mammals. The α, β and γ subunits of DmAMPK are more closely related to the mammalian homologues than to those of fungi or plants. DmAMPK and the rat homologue are activated by similar concentrations of AMP (half-maximal effect at 2-3µM), although the degree of stimulation of the Drosophila kinase was lower (4.5-fold compared with 22-fold). Both DmAMPK and the rat liver kinase were activated to much greater extents by AMP when they were purified by immunoprecipitation with the anti-PT172 antibody rather than other antibodies, and it was not possible to demonstrate any AMP dependence for DmAMPK after immunoprecipitation using the anti-QSSM antibody. The explanation for this curious behaviour remains unclear. The binding of the antibody to phospho-Thr-172 may produce a subtle conformational change that accentuates the effect of AMP, and this effect is perhaps mimicked by the T172D mutation (Pan, 2002).

In several respects the biochemical properties of the insect system are closely related to those of the mammalian system. (1) Like mammalian AMPK, DmAMPK is allosterically activated by AMP, albeit to a lower extent. (2) Like the mammalian kinase, the insect kinase is activated by treatments that depleted cellular ATP and caused increases in AMP, such as oligomycin and hypoxia. This is associated with phosphorylation of Thr-184 within the activation loop, a regulatory feature exhibited by all AMPK/SNF1-related protein kinases. The present results also show that DmAMPK is activated by glucose deprivation, as are its homologues in budding yeast and mammalian cells. (3) The phosphorylation of acetyl-CoA carboxylase by AMPK at a homologous site near the N-terminus (Ser-79/Ser-93) is also conserved between mammals and insects (Pan, 2002).

The results strongly suggest that the putative α, β and γ subunit sequences identified by homology with the mammalian homologues do indeed correspond to the subunits of a heterotrimeric DmAMPK complex in Drosophila cells. In the case of the α subunit, it was possible to immunoprecipitate kinase activity detectable using the SAMS peptide (a rather specific substrate for mammalian AMPK) with either of two antibodies (anti-CQSS and anti-PT172) made against synthetic peptides derived from the sequence. The detection of the 60kDa α subunit by Western blotting with the anti-PT172 antibody after oligomycin treatment, as well as phosphorylation of Ser-93 on DmACC, was also reduced or abolished by treatment with dsRNA targeted against the putative β or γ subunits, as well as dsRNA targeted against the α subunit itself. This provides evidence that all three subunits are required to form a functional complex in insect cells. Very similar findings have been reported in the mammalian and yeast systems. In mammals, significant expression of the recombinant α subunit is not seen unless DNAs encoding a β and γ subunit are co-transfected with that encoding the α subunit. In budding yeast, disruption of the genes encoding the γ subunit (SNF4), or those encoding all three β subunits (SIP1, SIP2, GAL83) results in the same phenotype as disruption of the gene encoding the catalytic subunit, SNF1 (Pan, 2002).

Using the anti-PT172 antibody, active DmAMPK appears to be largely confined to the nucleus of Dmel2 cells. In that respect, it is similar to the α2 isoform of mammalian AMPK, which is located in the nucleus in the pancreatic β cell line, INS-1, as well as in neurons in the hippocampus and cortex of rat brain, and in skeletal muscle. In budding yeast, the Gal83p isoform of the β subunit appears to target the SNF1 complex to the nucleus. It should be noted that the anti-PT172 antibody detects only the phosphorylated, activated form of DmAMPK-α, and the possibility cannot be ruled out that there is a pool of inactive kinase in the cytoplasm. As expected, the nuclear fluorescence obtained with the anti-PT172 antibody is greatly enhanced if the cells had been treated with oligomycin. This difference, together with the abolition of the signal in cells pre-treated with dsRNA targeted at the α subunit, confirmed that the antibody is specific for the phosphorylated α subunit of DmAMPK (Pan, 2002).

AMP-activated protein kinase phosphorylates retinoblastoma protein to control mammalian brain development

AMP-activated protein kinase (AMPK) is an evolutionarily conserved metabolic sensor that responds to alterations in cellular energy levels to maintain energy balance. While its role in metabolic homeostasis is well documented, its role in mammalian development is less clear. This study demonstrates that mutant mice lacking the regulatory AMPK β1 subunit have profound brain abnormalities. The β1^−/− mice show atrophy of the dentate gyrus and cerebellum, and severe loss of neurons, oligodendrocytes, and myelination throughout the central nervous system. These abnormalities stem from reduced AMPK activity, with ensuing cell cycle defects in neural stem and progenitor cells (NPCs). The β1^−/− NPC deficits result from hypophosphorylation of the retinoblastoma protein (Rb), which is directly phosphorylated by AMPK at Ser⁸⁰⁴. The AMPK-Rb axis is utilized by both growth factors and energy restriction to increase NPC growth. These results reveal that AMPK integrates growth factor signaling with cell cycle control to regulate brain development (Dasgupta, 2009).

Search PubMed for articles about Drosophila Ampk

Alessi, D. R., Sakamoto, K. and Bayascas, J. R. (2006). Lkb1-dependent signaling pathways. Annu. Rev. Biochem. 75: 137-163. PubMed Citation: 16756488

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Borg, J. P., et al. (1998). The X11 protein slows cellular amyloid precursor protein processing and reduces Aß40 and Aßß2 secretion. J. Biol. Chem. 273: 14761-14766. 9614075

Culmsee, C., Monnig, J., Kemp, B. E. and Mattson, M. P. (2001). AMP-activated protein kinase is highly expressed in neurons in the developing rat brain and promotes neuronal survival following glucose deprivation. J. Mol. Neurosci. 17: 45-58. PubMed Citation: 11665862

Dasgupta, B. and Milbrandt, J. (2007). Resveratrol stimulates AMP kinase activity in neurons. Proc. Natl. Acad. Sci. 104: 7217-7222. PubMed Citation: 17438283

Dasgupta, B. and Milbrandt, J. (2009). AMP-activated protein kinase phosphorylates retinoblastoma protein to control mammalian brain development. Dev. Cell 16(2): 256-70. PubMed Citation: 19217427

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Hardie, D. G. (2007b). AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy. Nat. Rev. Mol. Cell Biol. 8: 774-785. PubMed Citation: 17712357

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Lee, J. H., et al. (2010). Sestrin as a feedback inhibitor of TOR that prevents age-related pathologies. Science 327: 1223-1228. PubMed Citation: 20203043

Levine, B. and Kroemer, G. (2008). Autophagy in the pathogenesis of disease. Cell 132: 27-42. PubMed Citation: 18191218

Liang, J., et al. (2007). The energy sensing LKB1-AMPK pathway regulates p27(kip1) phosphorylation mediating the decision to enter autophagy or apoptosis. Nat. Cell Biol. 9: 218-224. PubMed Citation: 17237771

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Mandal, S., Guptan, P., Owusu-Ansah, E., Banerjee, U. (2005). Mitochondrial regulation of cell cycle progression during development as revealed by the tenured mutation in Drosophila. Dev. Cell 9(6): 843-54. PubMed Citation: 16326395

McCullough, L. D., et al. (2005). Pharmacological inhibition of AMP-activated protein kinase provides neuroprotection in stroke. J. Biol. Chem. 280: 20493-20502. PubMed Citation: 15772080

Medina, P. M., et al. (2006). A novel forward genetic screen for identifying mutations affecting larval neuronal dendrite development in Drosophila melanogaster. Genetics 172: 2325-2335. PubMed Citation: 16415365

Mirouse, V., Swick, L. L., Kazgan, N., St Johnston, D. and Brenman, J. E. (2007). LKB1 and AMPK maintain epithelial cell polarity under energetic stress. J. Cell Biol. 177(3): 387-92. PubMed Citation: 17470638

Norga, K. K., et al. (2003). Quantitative analysis of bristle number in Drosophila mutants identifies genes involved in neural development. Curr. Biol. 13: 1388-1396. PubMed Citation: 12932322

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date revised: 3 August 2009

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