BIOLOGICAL OVERVIEW

Phosphorylation is the main mechanism by which the cell transfers information from protein to protein along a signal transduction cascade. In the phosphorylation process, a phosphate residue is attached by means of an enzyme termed a kinase, to an amino acid residue of a downstream protein. Zeste-white 3, also known as Shaggy, is one of crucial kinases in the cell, and a critical one at that. Because for a long time, the Wingless receptor had not been identified, much research has focused on Shaggy, since Shaggy tranduces the wingless signal in the cell. Thus it is important to understand the place of Shaggy in the wingless signal transduction cascade.

What is the target of Shaggy phosphorylation? Wingless, through its receptor, inactivates shaggy. Genetic epistasis tests place shaggy upstream of armadillo (Peifer, 1994a and Sigfried, 1994). Mutations in shaggy mimic effects of the Wingless signal, resulting in an accumulation of high levels of cytoplasmic Arm in all cells (Peifer, 1994a). Together Shaggy/Zeste-white 3, and two other proteins, Axin and APC are termed the "ß-catenin destruction complex," which in Drosophila is responsible for degrading Armadillo, the ß-catenin homolog, thus releasing Pangolin, which subsequently enters the nucleus to activate Wingless target genes,

ARM is an integral part of the adherens junction as is ARM's closest vertebrate homolog, beta-catenin. The adherens junction is an adhesive contact point between cells, maintained by proteins that have connections across the membrane to the inside of the cell. Phosphorylation of ARM stabilizes its cytoplasmic form, where it may interact with other signal transduction molecules to pass the wingless signal into the nucleus (Peifer, 1994b). dishevelled and porcupine act upstream of shaggy. In porcupine mutants, Wingless appears confined to wingless expressing cells (Siegfried, 1994). Thus porcupine may be involved in Wingless secretion. dishevelled mutation adds no greater phenotypic effect to shaggy mutants, suggesting that dishevelled is upstream of shaggy, perhaps passing the signal from the Wingless receptor to the Shaggy kinase (Siegfried, 1994). Thus Shaggy is between the Dishevelled and Armadillo in the signal transduction pathway, receiving signals from Dishevelled and modifying Armadillo's association with junctions and its signaling process to the nucleus.

A model is presented for the role of Axin in Wnt signal transduction. In an unstimulated cell, GSK-3beta is active and phosphorylates Axin, which in turn, recruits beta-catenin into the Axin/GSK-3beta complex. By virtue of its proximity to GSK-3beta, beta-catenin is then phosphorylated. Phosphorylated beta-catenin is then targeted for degradation. Upon transduction of the Wnt signal through the Frizzled (Fz) receptors to Dishevelled, GSK-3beta kinase activity is inhibited so that PP2A (see Drosophila Twins) dephosphorylates Axin. Unphosphorylated Axin, in turn, no longer recruits beta-catenin to the complex. Failure of beta-catenin to associate with the Axin/GSK-3beta complex prevents its phosphorylation by GSK-3beta so that it can accumulate to high levels in the cytoplasm and nucleus and activate transcription in concert with the Tcf/Lef-1 family of transcription factors (Pangolin in Drosophila). GSK-3beta also phosphorylates APC, which may facilitate beta-catenin recruitment into the complex; however, this event has not been shown to be regulated by Wnt signaling (Willert, 1999).

Shaggy also appears to fill a 'neurogenic' role. At the site where neural precursors develop, achaete and scute are initially expressed in a group of equivalent cells (the proneural cluster). shaggy appears to act downstream of Notch for transduction of an inhibitory signal to adjacent cells. This second role of shaggy, now virtually ignored because of the current preoccupation with the function of Suppressor of hairless, may be due to modulation of some component of the Notch pathway (Simpson, 1993).

Work from Xenopus has clarified the interaction between Xgsk-3 (Shaggy in Drosophila) and ß-catenin (Armadillo in Drosophila). Xgsk-3 phosphorylates a ß-catenin serine/threonine rich site, which is conserved in the Drosophila protein Armadillo and in plakoglobin (a member of the same protein family). ß-catenin mutants lacking this site are more active in inducing an ectopic axis in Xenopus embryos and are more stable than wild-type ß-catenin in the presence of Xgsk-3 activity, supporting the hypothesis that Xgsk-3 is a negative regulator of ß-catenin, acting through the amino-terminal phosphorylation site. Xgsk-3 functions to destabilize ß-catenin and thus decrease the amount of ß-catenin available for signaling. The levels of endogenous ß-catenin in the nucleus increase in the presence of the dominant-negative Xgsk-3 mutant, suggesting that a role of Xgsk-3 is to regulate the steady-state levels of ß-catenin within specific subcellular compartments (Yost, 1996).

Work in the mouse suggests that protein kinase C (see Drosophila Protein kinase C) functions upstream of Shaggy. In mouse 10T1/2 fibroblasts, the activity of glycogen synthase kinase-3 (GSK-3), the murine homolog of Zw3/Sgg, is inactivated by Wingless. This occurs through a signaling pathway that is distinct from the insulin-mediated regulation of GSK-3, because Wg signaling to GSK-3 is insensitive to wortmannin. Wg-induced inactivation of GSK-3 is sensitive to both the protein kinase C (PKC) inhibitor Ro31-8220 and prolonged pre-treatment of 10T1/2 fibroblasts with phorbol ester. These findings provide the first biochemical evidence in support of the genetically defined pathway from Wg to Zw3/Sgg, and suggest a previously uncharacterized role for a PKC upstream of GSK-3/Zw3 during Wnt/Wg signal transduction (Cook, 1996).

The proposal that the Wingless signal is mediated by repression of Shaggy kinase activity was tested by overexpressing zeste white 3 in a tissue-specific fashion using the UAS/GAL4 binary expression system. Wild-type zw3 cDNA was placed under transcriptional control of the yeast GAL4 upstream activating sequence (UAS). UAS-zw3 flies were mated to flies that express the yeast transcriptional activator GAL4 in either a cell- or tissue-specific fashion to drive chronic expression of zw3. Elevated levels of zeste white 3 in the ectoderm and mesoderm result in phenotypes that resemble a loss of wingless. Overexpression of zeste white 3 in the mesoderm disrupts several Wingless-dependent processes, including the specification of a unique cell type in the larval midgut (the copper cell), the formation of the second midgut constriction, and the expression of Wingless target genes Ultrabithorax and decapentaplegic in the mesoderm, and labial in the endoderm. Interstitial cells normally found interspersed with the copper cells are still present. This loss of copper cells is similar to the phenotypes observed due to a loss of labial expression or wg expression, both required for the specification of the copper cells. The second midgut constriction is dependent on Wg signaling; in wg, dishevelled, or armadillo mutant embryos, this constriction does not form. Interestingly, in zw3 mutant embryos the second midgut constriction does form, but it is abnomal, appearing to have multiple folds. Zeste white 3 regulates the stability of Armadillo, which is essential for transducing the Wingless signal to the nucleus. zeste white 3 overexpression blocks Wingless signaling through the modulation of Armadillo since expression of a constitutively active form of Armadillo, which is independent of Zeste white 3 regulation, is epistatic to overexpression of zeste white 3 (Seitz, 1998).

PP2A and GSK-3β act antagonistically to regulate active zone development

The synapse is composed of an active zone apposed to a postsynaptic cluster of neurotransmitter receptors. Each Drosophila neuromuscular junction comprises hundreds of such individual release sites apposed to clusters of glutamate receptors. This study shows that protein phosphatase 2A (PP2A) is required for the development of structurally normal active zones opposite glutamate receptors. When PP2A is inhibited presynaptically, many glutamate receptor clusters are unapposed to Bruchpilot (Brp), an active zone protein required for normal transmitter release. These unapposed receptors are not due to presynaptic retraction of synaptic boutons, since other presynaptic components are still apposed to the entire postsynaptic specialization. Instead, these data suggest that Brp localization is regulated at the level of individual release sites. Live imaging of glutamate receptors demonstrates that this disruption to active zone development is accompanied by abnormal postsynaptic development, with decreased formation of glutamate receptor clusters. Remarkably, inhibition of the serine-threonine kinase GSK-3beta completely suppresses the active zone defect, as well as other synaptic morphology phenotypes associated with inhibition of PP2A. These data suggest that PP2A and GSK-3beta function antagonistically to control active zone development, providing a potential mechanism for regulating synaptic efficacy at a single release site (Viquez, 2009).

This study demonstrates that the serine-threonine phosphatase PP2A is required in the presynaptic neuron for normal development and maturation of presynaptic release sites. This action of PP2A is opposed by the serine-threonine kinase GSK-3β, suggesting that this phosphatase/kinase pair co-regulate the phosphorylation state and activity of proteins that are required for proper synaptic development (Viquez, 2009).

At the Drosophila NMJ, the synaptic terminal of a motoneuron is a branched chain of synaptic boutons whose gross structure is strongly influenced by the cytoskeleton. Within each synaptic terminal, there are hundreds of individual synapses, neurotransmitter release sites with an active zone directly apposed to a cluster of postsynaptic glutamate receptors. Most studies in Drosophila have focused on genes controlling synaptic terminal development. However with the recent development of antibodies to the active zone component Bruchpilot and the essential glutamate receptor DGluRIII, a genetic analysis of active zone and postsynaptic density development is now feasible. Previous studies have demonstrated that PP2A acts in the motoneuron to control synaptic terminal morphology likely via regulation of microtubules. This study demonstrate that PP2A is also essential for the proper development of the individual synaptic unit, the active zone and glutamate receptor dyad (Viquez, 2009).

Presynaptic inhibition of PP2A impairs synaptic transmission, leading to a large decrease in quantal content. While investigating potential morphological explanations for defective transmitter release, it was observed that many glutamate receptor clusters are unapposed to the active zone protein Bruchpilot. This is not due to retraction of the presynaptic terminal, since apposed and unapposed GluR clusters are intermingled throughout the terminal in a salt and pepper pattern, and presynaptic structures such as synaptic vesicles are still apposed to the entire extent of the postsynaptic specialization. Instead, there is a defect at the level of the individual synapse. These GluR clusters may be unapposed to active zones, or may be apposed to abnormal active zones lacking Bruchpilot. Two lines of evidence suggest that these GluR clusters may be unapposed to active zones. First, Bruchpilot is required for the localization of T-bars to the active zone, so if many active zones are missing Bruchpilot, then there should be a decrease in the proportion of active zones with T-bars. However when PP2A is inhibited no change was seen in the proportion of active zones with T-bars. Second, with PP2A inhibition the number of Brp puncta is down, as is the density of active zones as defined by ultrastructural analysis. This suggests that there is not a large pool of active zones without Brp. Both of these findings suggest that there are fewer active zones, and that those active zones that form do contain Brp. If this is so, then why are GluR clusters present that are unapposed to active zones? This could be due either to a problem with synapse formation/maturation or maintenance. While it is not known which is the case, the model that there is a defect in the formation or maturation is preferred for the following reasons. First, unapposed receptors are more prevalent in the distal regions of the NMJ where new synapses tend to be added. Second, the unapposed receptors form quite small clusters, while newly forming GluR clusters in wild type are also quite small. Finally, live imaging reveals that fewer GluR clusters form late in larval development, demonstrating a defect in synapse formation (Viquez, 2009).

A model is proposed in which PP2A activity is required for the maturation phase of synapse development. In this view, at a wild type synapse a signal would initiate synapse formation, leading to postsynaptic clustering of glutamate receptors as well as transsynaptic interactions that form the tightly apposed pre- and postsynaptic membranes as seen in electron micrographs. Later, additional active zone components such as Brp would be recruited to the active zone, a process known to occur after GluR clustering. With PP2A inhibition, this unknown signal would still initiate synapse formation and induce GluR clusters. However, at some fraction of nascent synapses the maturation process would fail. The GluR clusters could be trapped in their small, immature state or lost, while the transsynaptic process leading to the tight apposition of pre- and postsynaptic membranes would also fail and Brp would not be recruited. The alternate model that synaptic maintenance is disrupted, and that unapposed GluR clusters are the remains of synapses at which the presynaptic terminal has been lost, cannot, however, be ruled out. Regardless of the precise mechanism, these data demonstrate that PP2A is required to ensure the correct apposition of structurally normal active zones and glutamate receptors at the synapse (Viquez, 2009).

PP2A is one of the major serine/threonine phosphatases in the cell, so inhibiting its function likely leads to hyperphosphorylation of many proteins. Hence, phenotypes could be due to the pleiotropic effects of misregulating numerous pathways. The data, however, argue for a good deal of specificity in the function of PP2A for the synaptic morphology phenotypes assayed. Inhibiting PP2A in the neuron leads to misapposed GluR clusters, a disrupted synaptic cytoskeleton, and an altered bouton morphology. Each of these phenotypes is suppressed when GSK-3β is inhibited. This suggests that these synaptic phenotypes are due to the misregulation of a pathway that is antagonistically regulated by PP2A and GSK-3β. Opposite phenotypes are not seen, however, when PP2A is overexpressed, suggesting that hyperphosphorylation affects this pathway more than hypophosphorylation. While genetic studies cannot prove that this phosphatase/kinase pair act directly on the same substrate, the simplest interpretation of the data is that PP2A and GSK-3β co-regulate the phosphorylation state and activity of a protein or proteins that are required for the proper development of active zones and the synaptic cytoskeleton. While these PP2A phenotypes are all suppressed by inhibition of GSK-3β, there is no suppression of the accumulation of synaptic material in the axon, a phenotype consistent with defects in axonal transport. Decreased transport of active zone material such as Brp is a plausible mechanism for the active zone defects in this mutant. However, the failure of GSK-3β inhibition to suppress the axonal transport phenotype demonstrates that the active zone maturation and axon transport phenotypes are genetically separable. Hence, the accumulation of Brp in the axon cannot be responsible for the defects in synaptic maturation (Viquez, 2009).

The identity of the pathway regulated by PP2A and GSK-3β is not known. One candidate substrate is APC2, which binds to and stabilizes the plus end of microtubules and which is a characterized substrate of both PP2A and GSK-3β. In hippocampal cells phosphorylation of APC by GSK-3β inhibits APC function and so disrupts microtubule stability and axon outgrowth. It was shown that loss of APC2 dominantly enhances the PP2A phenotype, which is consistent with the model from hippocampal cells that phosphorylating APC decreases its function. However, if APC2 were the key substrate, then it would be predicted that homozygous APC2 mutants, where all APC2 function is lost, should replicate the PP2A phenotype. However a synaptic apposition phenotype is not seen in recessive mutants for APC2 or in APC1/APC2 double mutants. Instead, the enhancement of the PP2A phenotype by the loss of APC2 suggests that APC2 promotes PP2A function, possibly in its role as a scaffolding molecule. Wnt signaling is candidate pathway for mediating these synaptic phenotypes because wnt signaling is required for normal Drosophila NMJ development and because GSK-3β and PP2A regulate the phosphorylation state of β-catenin in canonical wnt signaling. Inhibition of PP2A would be predicted to lead to hyperphosphorylation and destruction of β-catenin, thereby blocking wnt signaling. However it is unlikely that the PP2A synaptic phenotype is due to loss of canonical wnt signaling. First, this study found that expression of a constitutively active β-catenin does not suppress the PP2A synaptic phenotype but instead has a slight tendency to enhance the cytoskeletal defect. Second, APC functions as part of the destruction complex that leads to degradation of β-Catenin and block of wnt signaling, however APC mutants enhance rather than suppress the PP2A phenotype. These results are inconsistent with the model that the phenotype is due to decreased canonical wnt signaling through β-Catenin. However, the data are consistent with a role for β-catenin-independent wnt signaling. A third candidate substrate is Futsch, since it can be phosphorylated by GSK-3β and the effect of reduction of PP2A activity on Futsch structure is suppressed by reduction in GSK-3β levels. Continued genetic analysis may lead to the identification of the relevant substrate(s) that are antagonistically regulated by PP2A and GSK-3β to control synaptic development (Viquez, 2009).

There are interesting parallels between the function of PP2A and GSK-3β in the developing Drosophila neuromuscular system and in the pathogenesis of neurodegenerative diseases such as Alzheimer's. In Drosophila, PP2A antagonizes GSK-3β function to stabilize the synaptic cytoskeleton and promote synapse formation. In models of Alzheimer's Disease, PP2A and GSK-3β also act antagonistically, for example in regulating the phosphorylation state of tau. In addition, disruptions to the axonal cytoskeleton and synapse loss are early events in Alzheimer's pathogenesis. Characterizing the function of PP2A/GSK-3β in regulating cytoskeletal and synaptic integrity during development may provide insights into their role in regulating cytoskeletal and synaptic integrity during disease (Viquez, 2009).

Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo

Genetic studies in Drosophila reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR) pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches this study tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways. Biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-β (GSK3β) a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3β activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia. This study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At the biochemical level it was found that both these pathways converge at GSK3β to control Myc protein stability, while genetic analysis shows that insulin and TOR pathways have different requirements for Myc activity during development of the eye, suggesting that Myc might be differentially induced by these pathways during growth or proliferation of cells that make up the ommatidia (Parisi, 2011).

Previous studies in vertebrates have indicated a critical function for Myc downstream of growth factor signaling including insulin-like growth factor, insulin and TOR pathways. In Drosophila, despite a few notes that Myc transcriptional activity acts downstream of insulin and TOR pathways, no clear molecular mechanisms linking these pathways to Myc have been elucidated yet (Parisi, 2011).

It has been demonstrated that inhibition of GSK3β prevents Myc degradation by the proteasome pathway. This study further unravels the pathways that control Myc protein stability and shows that signaling by insulin and TOR induce Myc protein accumulation by regulating GSK3β activity in S2 cells. GSK3β is a constitutively active kinase that is regulated by multiple signals and controls numerous cellular processes. With the biochemical data it is proposed that GSK3β acts as a common point where insulin and TOR signaling converge to regulate Myc protein stability (see Model showing the proposed relationship between Myc and the insulin and TOR signaling pathways). In particular, activation of insulin signaling was shown to induce activation of Akt, an event that is accompanied by GSK3β phosphorylation on Ser 9 that causes its inactivation and Myc protein to stabilize. Interestingly, insulin-induced Myc protein accumulation, when GSK3β activity was blocked by the presence of LiCl or by expression of GSK3β-KD, was similar to that obtained with insulin alone. Since it was shown that activation of insulin signaling leads to GSK3β inhibition and to an increase in Myc protein, if insulin and GSK3β signaling were acting independently, it would be expected that activation of insulin signaling concomitantly with the inhibition of GSK3β activity would result in a higher level of Myc than that obtained with insulin or LiCl alone. The results instead showed a similar level of Myc protein accumulation with insulin in the presence of GSK3β inhibitors as compared to insulin alone, supporting the hypothesis that GSK3β and insulin signaling, at least in the current experimental condition, depend on each other in the mechanism that regulates Myc protein stability (Parisi, 2011).

In a similar biochemical approach, the effect of AAs was analyzed on Myc protein stability and how TOR signaling is linked to mechanisms that inactivate GSK3β to stabilize Myc protein in S2 cells. In these experiments it was possible to demonstrate that treatment with amino acids (AAs) increased Myc protein stability, and it was also shown that treatment with rapamycin, an inhibitor of TORC1, reduced insulin-induced Myc upregulation. The reduction of Myc protein accumulation by rapamycin was blocked by inhibition of the proteasome pathway, linking TOR signaling to the pathway that controls Myc protein stability. TORC1 is a central node for the regulation of anabolic and catabolic processes and contains the central enzyme Rheb-GTPase, which responds to amino acids by activating TOR kinase to induce phosphorylation of p70-S6K and 4E-BP1. Analysis of the molecular mechanisms that act downstream of TOR to regulate Myc stability shows that AA treatment induces p70-S6K to phosphorylate GSK3β on Ser 9, an event that results in its inactivation and accumulation of Myc protein (Parisi, 2011).

Reducing GSK3β activity with LiCl, in medium lacking AAs, resulted in a slight increase in Myc protein levels. Adding back AAs lead to a substantial increase in Myc protein levels, which did not further increase when AAs where added to cells in the presence of the GSK3β inhibitor LiCl. These events were accompanied by phosphorylation of S6K on Thr 398, which correlated with phosphorylation of GSK3β on Ser 9. From these experiments it is concluded that TOR signaling also converges to inhibit GSK3β activity to regulate Myc protein stability. However, it has to be pointed out that since AAs alone increased Myc protein levels to a higher extent than that observed with LiCl alone, the experiments also suggest that Myc protein stability by TOR signaling is not solely directed through the inhibition of GSK3β activity, but other events and/or pathways contribute to Myc regulation. In conclusion, the biochemical experiments demonstrate that GSK3β acts downstream of insulin and TOR pathways to control Myc stability, however it cannot be excluded that other pathways may control Myc protein stability upon insulin and amino acids stimulation in S2 cells (Parisi, 2011).

Reduction of insulin and TOR signaling in vivo reduces cell size and proliferation, and clones mutant for chico, the Drosophila orthologue of IRS1-4, or for components of TOR signaling, are smaller due a reduction in size and the number of cells. The experiments showed that reducing insulin signaling by expression of PTEN or using TOR^TED, a dominant negative form of TOR, decreased Myc protein levels in clones of epithelial cells of the wing imaginal discs, while the opposite was true when these signals were activated using Dp110 or Rheb^AV4 . Those experiments suggested that the mechanism of regulation of Myc protein by insulin and TOR pathways was conserved also in vivo in epithelial cells of the larval imaginal discs (Parisi, 2011).

During these experiments it was also noted that Myc protein was induced in the cells surrounding and bordering the clones (non-autonomously), particularly when clones where positioned along the dorsal-ventral axis of the wing disc. This upregulation of Myc protein was not restricted to components of the insulin signaling pathway since it was also observed in cells surrounding the clones mutant for components of the Hippo pathway or for the tumor suppressor lethal giant larvae (lgl), which upregulates Myc protein cell-autonomously. It is suspected that this non-autonomous regulation of Myc may be induced by a novel mechanism that controls proliferation of cells when 'growth' is unbalanced. It can be speculated that clones with different growth rates, caused by different Myc levels, might secrete factors to induce Myc expression in neighboring cells. As a consequence, these Myc-expressing cells will speed up their growth rate in an attempt to maintain proliferation and tissue homeostasis. Further analysis is required to identify the mechanisms responsible for this effect (Parisi, 2011).

In order to distinguish if Myc activity was required downstream of insulin and TOR signaling to induce growth, a genetic analysis was performed. The ability to induce growth and proliferation was measured in the eye by measuring the size and number of the ommatidia from animals expressing members of the insulin and TOR pathways in different dm genetic background (dm⁺, dm^P0 and dm⁴). The data showed that Dp110 increased the size and number of the ommatidia, however only the alteration in the total number was dependent on dm levels. These data suggest that Myc is required downstream of insulin pathway to achieve the proper number of ommatidia. However, when insulin signaling was reduced by PTEN, a significant decrease in the size of ommatidia was seen and it was dependent on dm expression levels, suggesting that Myc activity is limiting for ommatidial size and number. Activation of TOR signaling induces growth, and the genetic analysis showed that Myc significantly contributes to the size of the ommatidial cells thus suggesting that Myc acts downstream of TOR pathway to control growth (Parisi, 2011).

Recent genomic analysis showed a strong correlation between the targets of Myc and those of the TOR pathway, implying that they may share common targets. In support of this observation, mosaic analysis with a repressible cell marker (MARCM) experiments in the developing wing disc showed that overexpression of Myc partially rescues the growth disadvantage of clones mutant for the hypomorphic Rheb^7A1 allele, further supporting the idea that Myc acts downstream of TOR to activate targets that control growth in these clones (Parisi, 2011).

The genetic interaction revealed a stronger dependence on Myc expression when Rheb was used as opposed to S6K. A possible explanation for this difference could lie in the fact that S6K is not capable of auto-activation of its kinase domain unless stimulated by TOR kinase. TOR activity is dependent on its upstream activator Rheb; consequently the enzymatic activity of the Rheb/GTPase is the limiting factor that influences S6K phosphorylation and therefore capable of maximizing its activity (Parisi, 2011).

Interestingly, these experiments also showed that activation of TOR signaling has a negative effect on the number of ommatidia, and this correlates with the ability of Rheb^AV4 to induce cell death during the development of the eye imaginal disc. Rheb-induced cell death was rescued in a dm^P0 mutant background, which leading to the speculation that 'excessive' protein synthesis, triggered by overexpression of TOR signaling, could elicit a Myc-dependent stress response, which induces apoptosis. Alternatively, high protein synthesis could result in an enrichment of misfolded proteins that may result in a stress response and induces cell death. Further analysis is required to delineate the mechanisms underlying this process (Parisi, 2011).

This analyses provide novel genetic and biochemical evidences supporting a role for Myc in the integration of the insulin and TOR pathway during the control of growth, and highlights the role of GSK3β in this signaling. It was found that insulin signaling inactivates GSK3β to control Myc protein stability, and a similar biochemical regulation is also shared by activation of the TOR pathways. In support of this data, a recent genomic analysis in whole larvae showed a strong correlation between the targets of Myc and those of the TOR pathway; however, less overlap was found between the targets of Myc and those of PI3K signaling (Parisi, 2011).

Statistical analysis applied to the genetic interaction experiments revealed that, in the Drosophila eye, proliferation induced by activation of the insulin pathway is sensitive to variations in Myc levels, while a significant interaction was seen mostly when TOR increased cell size. The data therefore suggests that there is a correlation between Myc and the InR signaling and it is expected that the InR pathway also shares some transcriptional targets with Myc. Indeed, an overlap was found between the targets induced by insulin and Myc in Drosophila S2 cells and these targets have also been reported in transcriptome analyses in the fat body upon nutritional stress, suggesting that Myc acts downstream of InR/PI3K and TOR signaling and that this interaction might be specific to some tissues or in a particular metabolic state of the cell (Parisi, 2011).

Nebula/DSCR1 upregulation delays neurodegeneration and protects against APP-induced axonal transport defects by restoring calcineurin and GSK-3beta signaling

Post-mortem brains from Down syndrome (DS) and Alzheimer's disease (AD) patients show an upregulation of the Down syndrome critical region 1 protein (DSCR1), but its contribution to AD is not known. To gain insights into the role of DSCR1 in AD, this study explored the functional interaction between DSCR1 and the amyloid precursor protein (APP), which is known to cause AD when duplicated or upregulated in DS. The Drosophila homolog of DSCR1, Nebula, was found to delay neurodegeneration and ameliorates axonal transport defects caused by APP overexpression. Live-imaging reveals that Nebula facilitates the transport of synaptic proteins and mitochondria affected by APP upregulation. Furthermore, Nebula upregulation was shown to protect against axonal transport defects by restoring calcineurin and GSK-3beta signaling altered by APP overexpression, thereby preserving cargo-motor interactions. As impaired transport of essential organelles caused by APP perturbation is thought to be an underlying cause of synaptic failure and neurodegeneration in AD, these findings imply that correcting calcineurin and GSK-3beta signaling can prevent APP-induced pathologies. The data further suggest that upregulation of Nebula/DSCR1 is neuroprotective in the presence of APP upregulation and provides evidence for calcineurin inhibition as a novel target for therapeutic intervention in preventing axonal transport impairments associated with AD (Shaw, 2013).

Although upregulation of APP had been shown to negatively influence axonal transport in mouse and fly models, mechanisms by which APP upregulation induces transport defects are poorly understood. Several hypotheses have been proposed, including titration of motor/adaptor by APP, impairments in mitochondrial bioenergetics, altered microtubule tracks, or aberrant activation of signaling pathways. The motor/adaptor titration theory suggests that excessive APP-cargos titrates the available motors away from other organelles, thus resulting in defective transport of pre-synaptic vesicles. The finding that Nebula co-upregulation enhanced the movement and delivery of both synaptotagmin and APP to the synaptic terminal argues against this hypothesis. In addition, earlier findings suggest that Nebula upregulation alone impaired mitochondrial function and elevated ROS level, thus implying that Nebula is not likely to rescue APP-dependent phenotypes by selectively restoring mitochondrial bioenergetics. Furthermore, consistent with a recent report showing normal microtubule integrity in flies overexpressing either APP-YFP or activated GSK-3βM (Weaver, 2013), the data revealed normal gross microtubule structure in flies with APP overexpression. Together, these results suggest that changes in gross microtubule structure and stability is not a likely cause of APP-induced transport defects (Shaw, 2013).

Instead, the current results support the idea that Nebula facilitates axonal transport defects by correcting APP-mediated changes in phosphatase and kinase signaling pathways. First, APP upregulation was found to elevate intracellular calcium level and calcineurin activity, and restoring calcineurin activity to normal suppresses the synaptotagmin aggregate accumulation in axons. The observed increase in calcium and calcineurin activity is consistent with reports of calcium dyshomeostasis and elevated calcineurin phosphatase activity found in AD brains, as well as reports demonstrating elevated neuronal calcium level due to APP overexpression and increased calcineurin activation in Tg2576 transgenic mice carrying the APP^swe mutant allele. Second, APP upregulation resulted in calcineurin dependent dephosphorylation of GSK-3β at Ser9 site, a process thought to activate GSK-3β kinase. APP upregulation also triggered calcineurin-independent phosphorylation at Tyr216 site, which has been shown to enhance GSK-3β activity. The kinase(s) that phosphorylates APP at Tyr216 is currently not well understood, it will be important to study how APP leads to Tyr216 phosphorylation in the future. Based on the current results, it is envisioned that APP overexpression ultimately leads to excessive calcineurin and GSK-3β activity, whereas nebula overexpression inhibits calcineurin to prevent activation of GSK-3β. The findings that nebula co-overexpression prevents GSK-3β activation and enhances the transport of APP-YFP vesicles are consistent with a recent report by Weaver (2013), in which it was found decreasing GSK-3β in fly increases the speed of APP-YFP movement. Furthermore, consistent with the current result that APP upregulation triggers GSK-3β enhancement and severe axonal transport defect, Weaver did not detect changes in GFP-synaptotagmin movement in the absence of APP upregulation (Shaw, 2013).

Active GSK-3β has been shown to influence the transport of mitochondria and synaptic proteins including APP, although the exact mechanism may differ between different cargos and motors. One mechanism proposed for GSK-3β-mediated regulation of axonal transport is through phosphorylation of KLC1, thereby disrupting axonal transport by decreasing the association of the anterograde molecular motor with its cargos. Accordingly, this study found that APP reduces KLC-synaptotagmin interaction while Nebula upregulation preserves it. Synaptotagmin transport in both the anterograde and retrograde directions are affected, consistent with previous reports showing that altering either the anterograde kinesin or retrograde dynein is sufficient affected transport in both directions. The results also support work suggesting that synaptotagmin can be transported by the kinesin 1 motor complex in addition to the kinesin 3/imac motor. As kinesin 1 is known to mediate the movement of both APP and mitochondria and phosphorylation of KLC had been shown to inhibit mitochondrial transport, detachment of cargo-motor caused by GSK-3β mediated phosphorylation of KLC may lead to general axonal transport problems as reported in this study. However, GSK-3β activation may also perturb general axonal transport by influencing motor activity or binding of motors to the microtubule tract. Interestingly, increased levels of active GSK-3β and phosphorylated KLC and dynein intermediate chain (DIC), a component of the dynein retrograde complex, have been observed in the frontal complex of AD patients. Genetic variability for KLC1 is thought to be a risk factor for early-onset of Alzheimer's disease. There is also increasing evidence implicating GSK-3β in regulating transport by modulating kinesin activity and exacerbating neurodegeneration in AD through tau hyperphosphorylation. It will be interesting to investigate if Nebula also modulates these processes in the future (Shaw, 2013).

SAlthough calcineurin had been shown to regulate many important cellular pathways, the link between altered calcineurin and axonal transport, especially in the context of AD, had not been established before. This study shows that calcineurin can regulate axonal transport through both GSK-3β independent and dependent pathways. This is supported by observation that the severity of the aggregate phenotype was worse for flies expressing APP and active calcineurin than it was for flies expressing APP and active GSK-3β. These findings point to a role for calcineurin in influencing axonal transport directly, perhaps through dephosphorylation of motor or adaptor proteins. The data also indicate that calcineurin in part modulates axonal transport through dephosphorylation of GSK-3β as discussed above; however, upregulation of APP is necessary for the induction of severe axonal transport problems, mainly by causing additional enhancement of GSK-3β signaling. GSK3 inhibition is widely discussed as a potential therapeutic intervention for AD; results suggest that perhaps calcineurin is a more effective target for delaying degeneration by preserving axonal transport (Shaw, 2013).

DSCR1 and APP are both located on chromosome 21 and upregulated in DS. Overexpression of DSCR1 alone had been contradictorily implicated in both conferring resistance to oxidative stress and in promoting apoptosis. Upregulation of Nebula/DSCR1 had also been shown to negatively impact learning and memory in fly and mouse models through altered calcineurin pathways. How could upregulation of DSCR1 be beneficial? It is proposed that DSCR1 upregulation in the presence of APP upregulation compensates for the altered calcineurin and GSK-3β signaling, shifting the delicate balance of kinase/phosphatase signaling pathways close to normal, therefore preserving axonal transport and delaying neurodegeneration. It is also proposed that axonal transport defects and synapse dysfunction caused by APP upregulation in the Drosophila model system occur prior to accumulation of amyloid plaques and severe neurodegeneration, similar to that described for a mouse model (Shaw, 2013).

DS is characterized by the presence of AD neuropathologies early in life, but most DS individuals do not exhibit signs of dementia until decades later, indicating that there is a delayed progression of cognitive declin. The upregulation of DSCR1 may in fact activate compensatory cell signaling mechanisms that provide protection against APP-mediated oxidative stress, aberrant calcium, and altered calcineurin and GSK3-β activity (Shaw, 2013).

GENE STRUCTURE

shaggy has multiple transcripts and is alternatively spliced. Of ten developmentally regulated transcripts, two seem to be responsible for early sgg activity. There are five different proteins generated by alternative splicing (Bourouis, 1990 and Ruel, 1993).

Length of genomic DNA - 40 kb

Exons - There are nine, whose translation products are incorporated in various combinations in the different protein transcripts.

PROTEIN STRUCTURE

Structural Domains

Shaggy has a catalytic domain found in serine/threonine specific protein kinases, linked to a region unusually rich in Gly, Ala and Ser (Bourouis, 1990).

Characterisation of protein isoforms encoded by the Drosophila Glycogen Synthase Kinase 3 gene shaggy

The Drosophila shaggy gene (sgg, GSK-3) encodes multiple protein isoforms with serine/threonine kinase activity and is a key player in diverse developmental signalling pathways. Currently it is unclear whether different Sgg proteoforms are similarly involved in signalling or if different proteoforms have distinct functions. This study used CRISPR/Cas9 genome engineering to tag eight different Sgg proteoform classes and determined their localization during embryonic development. Proteomic analysis was performed of the two major proteoform classes and mutant lines for both of these were generated for transcriptomic and phenotypic analysis. Distinct tissue-specific localization patterns were uncovered for all of the tagged proteoforms examined, most of which have not previously been characterised directly at the protein level, including one proteoform initiating with a non-standard codon. Collectively, this suggests complex developmentally regulated splicing of the sgg primary transcript. Further, affinity purification followed by mass spectrometric analyses indicate a different repertoire of interacting proteins for the two major proteoforms examined, one with ubiquitous expression (Sgg-PB) and one with nervous system specific expression (Sgg-PA). Specific mutation of these proteoforms shows that Sgg-PB performs the well characterised maternal and zygotic segmentations functions of the sgg locus, while Sgg-PA mutants show adult lifespan and locomotor defects consistent with its nervous system localisation. These findings provide new insights into the role of GSK-3 proteoforms and intriguing links with the GSK-3α and GSK-3β proteins encoded by independent vertebrate genes. This analysis suggests that different proteoforms generated by alternative splicing are likely to perform distinct functions (Korona, 2020).

date revised: 25 September 2023 

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