InteractiveFly: GeneBrief

alicorn: Biological Overview | References


Gene name - alicorn

Synonyms -

Cytological map position - 45A11-45A11

Function - signaling

Keywords - regulation of cellular energy homeostasis, maintains integrity of mature neurons under conditions of increased activity, cholesterol homeostasis, Appl processing, regulation of developmental and stress-induced autophagy

Symbol - alc

FlyBase ID: FBgn0260972

Genetic map position - 2R:5,022,880..5,025,455

Classification - AMP-activated protein kinase (AMPK) beta subunit glycogen binding domain

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

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 alcAd2 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 alcAd2 and alcδ12.125 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 norpAP24 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 norpAP24 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 GABAB 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).

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 AMPKG505 line, AMPK-null mutant lines, AMPKD1 and AMPKD2 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 (AMPKWT) allowed AMPK-null mutants to successfully develop into adults, the expression of kinase-dead AMPK (AMPKKR) 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 (AMPKTD), which is catalytically active even without phosphorylation by LKB1, was expressed in lkb1-null mutants. Remarkably, AMPKTD 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 (MRLCEE), whose regulatory phosphorylation site was mutated into phosphomimetic glutamates, was expressed in AMPK-GLC embryos. Strikingly, MRLCEE rescued the epithelial polarity defects caused by the loss of AMPK, and increased the percentage of cuticle-forming embryos from ~10% to ~30. MRLCEE 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 MRLCEE. 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 (AMPKDN) 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-1DN) failed to cause a block. More strikingly, human MRLCEE 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).

The neurodegeneration mutant löchrig interferes with cholesterol homeostasis and Appl processing

The novel Drosophila mutant löchrig (loe) 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 functional homology to the mammalian AMPK is supported further by the accumulation of fatty acids in the mutant, another pathway regulated by AMPK (Tschäpe, 2002).

AMPK negatively regulates HMG-CoA reductase, a key enzyme in cholesterol synthesis in vertebrates. In Drosophila, this protein is encoded by the columbus (clb) gene (Van Doren, 1998). To assess whether loe interacts with the clb mutation, flies were created homozygous for loe and heterozygous for two strong, embryonic lethal alleles of clb (which both had the same effect). clb/+; loe mutants show a weak but significant suppression of vacuolization compared with loe mutant flies. To confirm an interaction, lines expressing Clb were used in the loe background. In contrast to the clb mutation, Clb overexpression enhances the phenotype. Control flies, containing only the UAS-Clb construct but no neuronal promoter construct, did not differ from the original loe mutants. The interaction was quantified by counting holes in the different genotypes and measuring their total volume. The enhancement by Clb overexpression and suppression by the clb/+ mutant suggests that HMG-CoA reductase is negatively regulated by AMPK as in other organisms. In addition, an influence on the cholesterol ester level of loe was investigated. Overexpression of Clb slightly reduced, and introduction of one mutant copy of clb slightly increased, the cholesterol ester level in loe; however, the differences are not significant. Nevertheless, they are in agreement with the results on the neurodegenerative phenotype because the clb mutation suppresses and additional Clb enhances the phenotype. Interestingly, the function of HMG-CoA reductase in cholesterol synthesis is not conserved because insects cannot synthesize cholesterol de novo. However, many other downstream genes and regulatory feedback mechanisms are conserved, and one of them might connect HMG-CoA reductase and cholesterol ester (Tschäpe, 2002).

HMG-CoA reductase can be inhibited pharmacologically by a class of drugs called statins, which have also been shown to decrease the prevalence of Alzheimer's disease. To assess whether treatment with statins influences the neurodegeneration in loe, flies fed on glucose were compared with or without the drug lovastatin. Flies kept on lovastatin showed a suppression of the vacuolization compared with control animals. Treatment of wild-type flies with lovastatin revealed no adverse effects. These results show that the progressive neurodegeneration in loe can be slowed successfully by treatment with statins. The level of cholesterol ester was tested in loe flies treated with statins, but no significant difference could be found (Tschäpe, 2002).

Cholesterol homeostasis has been implicated in the processing of Aß from APP, as has statin treatment, which can dramatically decrease Aß production. Therefore, whether loe influences APPL, the fly homolog of human APP, was investigated. Appld mutants, which carry a deletion in the Appl gene, do not reveal any signs of neurodegeneration. However, crossing Appld with loe flies shows an enhancement of the loe vacuolization. The effect is weaker in loe flies carrying one copy of Appld (loe/loe; Appld/+) compared with homozygous double mutants (loe/loe; Appld/Appld) and can be detected in the central brain as well as the optic system (Tschäpe, 2002).

To determine whether loe might influence the APPL protein, Western blot analysis of brain extracts was performed. Using an anti-APPL polyclonal antibody, two bands were detected in w1118 flies, representing the genetic background used to induce the loe mutation. The bands correspond to the membrane-associated 145 kDa precursor and the 130 kDa secreted form, which are absent in Appld. In the loe mutant, similar amounts of APPL precursor protein are found; however, the level of the processed secreted form is reduced. Conversely, more of the secreted form is found when additional LoeI is expressed in neurons. This reveals a role for loe in APPL processing or stabilization of the processed form. To assess whether this effect is specific for APPL, the processing of Notch, which is cleaved by a mechanism similar to that of APP, was investigated. No differences were detected in the processing of Notch, suggesting a specific function of loe in APPL processing, possibly mediated by the X11alpha similarity domain. In addition, whether Columbus or statin treatment influences APPL processing in loe was investigated. Additional expression of Clb, which enhanced the neurodegenerative phenotype of loe, also enhances the processing effect, causing a slight further reduction of APPL processing. On the contrary, one copy of mutant clb or statin treatment slightly increased processing. This suggests that the neurodegenerative phenotype is correlated with the processing of APPL (Tschäpe, 2002).

Thus, a mutation in the AMPK gamma-subunit causes progressive neurodegeneration in Drosophila. AMPK is a central component of a protein kinase cascade conserved in eukaryotes that acts as a metabolic sensor to monitor the cellular AMP and ATP levels. In cases of ATP depletion, the major ATP function described to date is to activate energy-providing mechanisms while inactivating energy-consuming processes. AMPK is a heterotrimer, consisting of the catalytic alpha-subunit and the ß- and gamma-subunits, which are required for stabilization of the complex and kinase activity. The activity of the complex is regulated by phosphorylation through an upstream kinase, and both phosphorylation and dephosphorylation are sensitive to AMP levels. For all three subunits, different isoforms have been identified that assemble into specific AMPK complexes with distinguishable tissue distribution in peripheral tissues in vertebrates. Whereas most tissues predominantly express one gamma isoform, the human brain expresses three different isoforms. Interestingly, two of them have extended N-termini with no significant homology to each other, LoeI or any other protein. The loe mutation shows, for the first time, that such a brain-specific isoform has a unique function in brain maintenance, which cannot be substituted by other isoforms. This function probably goes beyond the basic role in energy regulation because all isoforms share the C-terminus, which is sufficient for a functional gamma-subunit and, therefore, a functional AMPK complex. It will be interesting to discover whether one of the human isoforms is also required specifically for neuronal survival (Tschäpe, 2002).

AMPK has a central role in cholesterol metabolism by regulating HMG-CoA reductase and hormone-sensitive lipase, which is involved in the breakdown of cholesterol ester in vertebrates. Although hormone-sensitive lipase has not been found in the brain, a cholesterol ester hydrolase activity is described for the brain; however, nothing is known about the potential regulation of this enzyme by AMPK. An inhibitory function of AMPK in the brain would lead to an overactivity of this hydrolase and, therefore, to a reduced level of cholesterol ester. A Drosophila protein with homology to hormone-sensitive lipase can be found in the Drosophila Sequencing Project, but unfortunately no mutant has been described so far. However, a deficiency deleting this enzyme was tested for genetic interactions with loe. Because this deficiency had no influence on the loe phenotype, it is assumed that it is not involved in the neurodegenerative phenotype. In contrast, this study shows a genetic as well as a pharmacologically induced interaction of loe with HMG-CoA reductase (clb). The interaction reveals that, as in vertebrates, AMPK acts upstream of HMG-CoA reductase. Because a mutation in clb suppresses and overexpression enhances the neurodegenerative loe phenotype, the inhibitory function of AMPK on HMG-CoA reductase seems to be conserved. Interestingly, the function of HMG-CoA reductase is not completely conserved between vertebrates and insects, because arthropods cannot synthesize cholesterol de novo. Rather, HMG-CoA reductase is involved in the production of non-sterol isoprenoids from mevalonate. The effect of HMG-CoA reductase on neurodegeneration cannot, therefore, be mediated through cholesterol synthesis and, as measurements show, the cholesterol level is unaltered in loe. However, the amount of cholesterol ester is lowered in loe and adding or removing Clb has a slight influence on it, and APPL processing in loe is influenced by Clb. In this context, it is worth mentioning that statins dramatically decrease Aß production before a reduction in cholesterol can be detected. This suggests that other members of the cholesterol pathway might regulate APP processing, possibly cholesterol ester (Tschäpe, 2002).

The loe mutation reveals a connection between cholesterol ester and progressive neurodegeneration in the model system Drosophila. In vertebrates, such a link has been established by the finding that accumulation of Aß can decrease cholesterol esterification in neurons (Koudinova, 1996; Liu, 1998). found that The level of cholesterol ester directly correlates with Aß production (Puglielli. 2001), and that elevated concentrations of cholesterol ester but not free cholesterol increase the generation of Aß. In contrast, it has been shown that lowering the cholesterol concentration inhibits APP cleavage by secretases and interferes with the localization of APP in membrane rafts (Simons, 1998; Frears, 1999). These are membrane microdomains consisting of lipids, proteins and cholesterol, and their correct composition seems to be required for normal APP processing. These results strengthen the likelihood of a role for cholesterol ester because the loe mutant links a reduced level of cholesterol ester, leaving free cholesterol unaltered, with decreased processing of APPL (Tschäpe, 2002).

These results clearly reveal a function of AMPK in APPL processing. However, the Appl mutant enhances the neurodegenerative phenotype of loe. Like knock-outs of APP in mice, the Appld null mutation of Drosophila displays only subtle neurological deficits. In the loe mutant background, however, Appl can be connected to progressive neurodegeneration, which might help to understand the function of APP proteins. Because the lack of APPL enhances the phenotype, this hints at a neuroprotective function, perhaps specifically of the soluble form of APPL -- this was also suggested by cell culture studies of APP. In this model, neurons would be more vulnerable to the effect of the loe mutation when APPL and its soluble form are missing. The Appl mutant itself might not show degeneration because the damaging event is absent (Tschäpe, 2002).

With the isolation of the loe mutant, a connection has been made between AMPK, a second enzyme (in addition to HMG-CoA reductase involved in cholesterol homeostasis) and neurodegeneration and APPL processing. This underlines the importance of the cholesterol biosynthesis pathway for the maintenance of the nervous system and for understanding of neurodegenerative diseases such as Alzheimer's. With the Drosophila loe mutant available, the role of this pathway in neurodegeneration can now be studied in an easily accessible model organism (Tschäpe, 2002).

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).

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).

AMPK and PPARδ agonists are exercise mimetics

The benefits of endurance exercise on general health make it desirable to identify orally active agents that would mimic or potentiate the effects of exercise to treat metabolic diseases. Although certain natural compounds, such as reseveratrol, have endurance-enhancing activities, their exact metabolic targets remain elusive. Therefore the effect of pathway-specific drugs on endurance capacities of mice were tested in a treadmill running test. PPARβ/δ agonist and exercise training synergistically increase oxidative myofibers and running endurance in adult mice. Because training activates AMPK and PGC1α, whether the orally active AMPK agonist AICAR might be sufficient to overcome the exercise requirement was tested. Unexpectedly, even in sedentary mice, 4 weeks of AICAR treatment alone induced metabolic genes and enhanced running endurance by 44%. These results demonstrate that AMPK-PPARδ pathway can be targeted by orally active drugs to enhance training adaptation or even to increase endurance without exercise (Narkar, 2008).

This study shows that the AMP-mimetic AICAR can increase endurance in sedentary mice by genetically reprogramming muscle metabolism in a PPARδ-dependent manner. A PPARδ agonist in combination with exercise synergistically induces fatigue-resistant type I fiber specification and mitochondrial biogenesis, ultimately enhancing physical performance. These changes correlate with an unexpected but interesting establishment of a muscle endurance gene signature that is unique to the drug-exercise paradigm. Such a signature is an outcome of molecular crosstalk and perhaps a physical association between exercise-activated AMPK and PPARδ. These findings identify a novel pharmacologic strategy to reprogram muscle endurance by targeting AMPK-PPARδ signaling axis with orally active ligands (Narkar, 2008).

Transgenic overexpression as well as knockout studies have identified PPARδ and AMPK as key regulators of type I fiber specification and endurance adaptations during exercise. Whether and how these endogenously expressed regulators can be targeted to reprogram adult muscle without exercise has been a subject of unresolved speculation. This study found that the AMPK activator AICAR increased oxygen consumption and endurance in untrained adult mice in part by stimulating PPARδ-dependent oxidative genes. Despite a demonstrated role for PPARδ in endurance, 4 week treatment with a potent and selective agonist failed to alter either fiber type composition or endurance, revealing that direct and pharmacologic activation of PPARδ is insufficient to enhance running performance. In contrast, transgenic overexpression of activated PPARδ at birth preprograms the nascent myofibers to transdifferentiate into slow-twitch fibers, thus imparting a high basal endurance capacity to adult transgenic mice. Apparently, once fiber type specification is complete in adults, the potential plasticity of muscle to synthetic activation of a single transcriptional pathway is constrained. Along these lines, the unexpected yet successful reprogramming of endurance in untrained adults with synthetic AMP-mimetic might be linked to the ability of AMPK to simultaneously target multiple transcriptional programs governed by its substrates such as PGC1α, PPARα and PPARδ, triggering a genetic effect akin to exercise (Narkar, 2008).

Interestingly, the recalcitrance of adult skeletal muscle endurance to manipulation by PPARδ agonist alone is relieved by combining drug treatment with exercise. Indeed, this strategy generates an endurance gene signature that is unique from either paradigm alone, reflecting a crosstalk between exercise and PPARδ signaling. Although exercise activates a cascade of signaling events, AMPK is thought to be central to this genetic adaptation for several reasons: (1) AMPK is a metabolic sensor that detects low ATP levels (such as occur during exercise) and in turn increases oxidative metabolism; (2) long-term effects of AMPK are in part mediated via regulation of gene expression; (3) exercise induces activation and nuclear import of AMPK, where it can potentially interact with transcription factors, and (4) transgenic mice defective for AMPK activation exhibit reduced voluntary exercise, making it an attractive exercise cue that modulates receptor signaling (Narkar, 2008).

The notion that exercise-activated AMPK interacts with PPARδ in regulating gene expression is supported by the demonstration that AMPK associates with PPARδ and dramatically increases basal and ligand-dependent transcription via the receptor. Despite physical interaction, it was found that AMPK does not induce PPARδ phosphorylation in metabolic labeling studies. Interestingly, AMPK and its previously reported substrate PGC1α synergistically increased PPARδ transcription, suggesting indirect regulation of receptor function by AMPK via coregulator modification. Nevertheless, the possible regulation of PPARδ by AMPK via direct protein-protein interaction cannot be ruled out. Indeed, regulation of other transcription factors by AMPK via similar mechanisms has been previously demonstrated. A physiological validation of AMPK-PPARδ interaction comes from the observation that GW1516 and AICAR (AMPK activator) synergistically induce several endurance-related genes in wild-type but not in PPARδ null primary muscle cells. More importantly, treatment of animals with AICAR and GW1516 creates a gene signature in skeletal muscle that replicates up to 40% of the genetic effects of combined exercise and GW1516 treatment. Notably, the shared genes between the two profiles are linked to oxidative metabolism, angiogenesis, and glucose sparing, pathways that are directly relevant to muscle performance (Narkar, 2008).

Although not all genes regulated by either exercise or exercise-PPARδ interaction are AMPK dependent, two key findings assign a critical role for the kinase in promoting endurance compared to other known exercise signals: (1) AMPK is constitutively active in VP16-PPARδ transgenic muscles that exhibit endurance without exercise; (2) AMPK activation by AICAR was sufficient to increase running endurance without additional exercise signals. Strikingly, the majority of the oxidative genes (30 out of 32) upregulated by AICAR are active in super-endurance VP16-PPARδ mice and perhaps are the core set of genes required to improve muscle performance. Interestingly, AICAR failed to induce oxidative gene expression in PPARδ null muscle cells, indicting the requirement of PPARδ, at least for regulation of oxidative metabolism by AMPK. Collectively, these findings demonstrate a molecular partnership between AMPK and PPARδ in reprogramming skeletal muscle transcriptome and endurance that can be readily exploited by orally active AMPK drugs to replace exercise (Narkar, 2008).

In humans, endurance exercise leads to physiological adaptations in the cardiopulmonary, endocrine, and neuromuscular systems. Although this investigation focused on skeletal muscle, extramuscular effects of PPARδ, AMPK, and exercise may also contribute to increased endurance. Although potentiation of extramuscular adaptations by PPARδ and AMPK agonists remains to be studied, it was found that drug treatment can reduce epididymal fat mass, possibly conferring additional systemic benefits. It is noteworthy that PPARδ is important for normal cardiac contractility, as well as for the endocrine function of adipose tissue. Similarly, the activation of AMPK by metformin is thought to mediate its ability to lower blood glucose levels. In addition to increasing performance in athletes, exercise has beneficial effects in a wide range of pathophysiological conditions, such as respiratory disorders, cardiovascular abnormalities, type 2 diabetes, and cancer risk. Therefore, understanding the effects of exercise on normal physiology and identifying pharmaceutically targetable pathways that can boost these effects is crucial. This study revealed that synthetic PPARδ activation and exercise -- and, more importantly, AMPK activation alone -- provide a robust transcriptional cue that reprograms the skeletal muscle genome and dramatically enhances endurance. It is believed that the strategy of reorganizing the preset genetic imprint of muscle (as well as other tissues) with exercise mimetic drugs has therapeutic potential in treating certain muscle diseases such as wasting and frailty as well as obesity where exercise is known to be beneficial (Narkar, 2008).


REFERENCES

Search PubMed for articles about Drosophila Alicorn

Borg, J. P., et al. (1998). The X11 protein slows cellular amyloid precursor protein processing and reduces Aß40 and Aß2 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 ID: 11665862

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

Dockendorff, T. C., et al. (2000). Genetic characterization of the 44D-45B region of the Drosophila melanogaster genome based on an F2 lethal screen. Mol. Gen. Genet. 263: 137-143. PubMed ID: 10732682

Dyck, J. R., et al. (1996). Regulation of 5'-AMP-activated protein kinase activity by the noncatalytic beta and gamma subunits. J. Biol. Chem. 271: 17798-17803. PubMed ID: 8663446

Edinger, A. L. and Thompson, C. B. (2004). Death by design: apoptosis, necrosis and autophagy. Curr. Opin. Cell Biol. 16: 663-669. PubMed ID: 15530778

Frears, E. R., et al. (1999). The role of cholesterol in the biosynthesis of ß-amyloid. Neuroreport 10: 1699-1705. 10501560

Gadalla, A. E., et al. (2004). AICA riboside both activates AMP-activated protein kinase and competes with adenosine for the nucleoside transporter in the CA1 region of the rat hippocampus. J. Neurochem. 88: 1272-1282. PubMed ID: 15009683

Hardie, D. G. (2003). Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status. Endocrinology 144(12): 5179-83. PubMed ID: 12960015

Hardie, D. G. and Frenguelli, B. G. (2007a). A neural protection racket: AMPK and the GABAB receptor. Neuron 53: 159-162. PubMed ID: 17224398

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

Hawley, S. A., et al. (2005). Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase. Cell. Metab. 2: 9-19. PubMed ID: 16054095

Hong, S. P., et al. (2003). Activation of yeast Snf1 and mammalian AMP-activated protein kinase by upstream kinases. Proc. Natl. Acad. Sci. 100: 8839-8843. PubMed ID: 12847291

Ido, Y., Carling, D. and Ruderman, N. (2002). Hyperglycemia-induced apoptosis in human umbilical vein endothelial cells: inhibition by the AMP-activated protein kinase activation. Diabetes 51: 159-167. PubMed ID: 11756336

Koudinova, N. V., Berezov, T. T. and Koudinov, A. R. (1996). Multiple inhibitory effects of Alzheimer¬ís peptide Aß1-40 on lipid biosynthesis in cultured human HepG2 cells. FEBS Lett. 395: 204-206. 8898096

Kuramoto, N., et al. (2007). Phospho-dependent functional modulation of GABA(B) receptors by the metabolic sensor AMP-dependent protein kinase. Neuron 53: 233-247. PubMed ID: 17224405

Lee, J. H., et al. (2007). Energy-dependent regulation of cell structure by AMP-activated protein kinase. Nature 447(7147): 1017-20. PubMed ID: 17486097

Levine, B. and Kroemer, G. (2008). Autophagy in the pathogenesis of disease. Cell 132: 27-42. PubMed ID: 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 ID: 17237771

Lippai M, Csikos G, Maroy P, Lukacsovich T, Juhasz G, Sass M (2008) SNF4Agamma, the Drosophila AMPK gamma subunit is required for regulation of developmental and stress-induced autophagy. Autophagy 4: 476-486. PubMed ID: 18285699

Liu, Y., Peterson, D. A. and Schubert, D. (1998). Amyloid ß peptide alters intracellular vesicle trafficking and cholesterol homeostasis. Proc. Natl. Acad. Sci. 95: 13266-13271. 9789077

Lyman, R. F., Lawrence, F., Nuzhdin, S. V. and Mackay, T. F. (1996). Effects of single P-element insertions on bristle number and viability in Drosophila melanogaster. Genetics 143: 277-292. PubMed ID: 8722781

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

Narkar, V. A., et al. (2008). AMPK and PPARδ agonists are exercise mimetics. Cell 134: 405-415. PubMed ID: 18674809

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 ID: 12932322

Okoshi, R., et al. (2008). Activation of AMP-activated protein kinase induces p53-dependent apoptotic cell death in response to energetic stress. J. Biol. Chem. 283: 3979-3987. PubMed ID: 18056705

Pan, D. A. and Hardie, D. G. (2002). A homologue of AMP-activated protein kinase in Drosophila melanogaster is sensitive to AMP and is activated by ATP depletion. Biochem J 367: 179-186. PubMed ID: 12093363

Puglielli, L., et al. (2001). Acyl-coenzyme A: cholesterol acyltransferase modulates the generation of the amyloid ß-peptide. Nat. Cell Biol. 3: 905-912. PubMed ID: 11584272

Ramamurthy, S. and Ronnett, G. V. (2006). Developing a head for energy sensing: AMP-activated protein kinase as a multifunctional metabolic sensor in the brain. J. Physiol. (Lond) 574: 85-93. PubMed ID: 16690704

Russell, R. R. et al. (2004). AMP-activated protein kinase mediates ischemic glucose uptake and prevents postischemic cardiac dysfunction, apoptosis, and injury. J. Clin. Invest. 114: 495-503. PubMed ID: 15314686

Sanders, M. J., et al. (2007). Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade. Biochem. J. 403: 139-148. PubMed ID: 17147517

Scott, J. W., et al. (2004). CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations. J. Clin. Invest. 113: 274-284. PubMed ID: 14722619

Shaw, R. J., et al. (2004). The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc. Natl. Acad. Sci. 101: 3329-3335. PubMed ID: 14985505

Simons, M., et al. (1998). Cholesterol depletion inhibits the generation of ß-amyloid in hippocampal neurons. Proc. Natl. Acad. Sci. 95: 6460-6464. 9600988

Spasic, M. R., Callaerts, P. and Norga, K. K. (2008). Drosophila alicorn is a neuronal maintenance factor protecting against activity-induced retinal degeneration. J. Neurosci. 28: 6419-6429. PubMed ID: 18562613

Tschäpe, J. A., et al. (2002). The neurodegeneration mutant löchrig interferes with cholesterol homeostasis and Appl processing. EMBO J. 21: 6367-6376. 9600988

Turnley, A. M., et al. (1999). Cellular distribution and developmental expression of AMP-activated protein kinase isoforms in mouse central nervous system. J. Neurochem. 72: 1707-1716. PubMed ID: 10098881

Van Doren, M., Broihier, H. T., Moore, L. A. and Lehmann, R. (1998). HMG-CoA reductase guides migrating primordial germ cells. Nature 396: 466-469. 9853754

Woods, A., Salt, I., Scott, J., Hardie, D. G. and Carling, D. (1996). The alpha1 and alpha2 isoforms of the AMP-activated protein kinase have similar activities in rat liver but exhibit differences in substrate specificity in vitro. FEBS Lett. 397: 347-351. PubMed ID: 8955377

Woods, A., et al. (2003). LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr. Biol. 13: 2004-2008. PubMed ID: 14614828

Woods, A., et al. (2005). Ca2+/calmodulin-dependent protein kinase kinase-beta acts upstream of AMP-activated protein kinase in mammalian cells. Cell Metab. 2: 21-33. PubMed ID: 16054096


Biological Overview

date revised: 2 December 2008

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