shaggy: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - shaggy

Synonyms - zeste white 3

Cytological map position - 3B1

Function - signal transduction, enzyme

Keywords - segment polarity, wingless pathway, component of the ß-catenin destruction complex

Symbol - sgg

FlyBase ID:FBgn0003371

Genetic map position - 1-1.3

Classification - serine-threonine kinase - glycogen synthase kinase 3

Cellular location - cytoplasmic



NCBI link: Entrez Gene
sgg orthologs: Biolitmine
Recent literature
Wang, W., Li, J., Tan, J., Wang, M., Yang, J., Zhang, Z. M., Li, C., Basnakian, A. G., Tang, H. W., Perrimon, N. and Zhou, Q. (2021). Endonuclease G promotes autophagy by suppressing mTOR signaling and activating the DNA damage response. Nat Commun 12(1): 476. PubMed ID: 33473107
Summary:
Endonuclease G (ENDOG), a mitochondrial nuclease, is known to participate in many cellular processes, including apoptosis and paternal mitochondrial elimination, while its role in autophagy remains unclear. This study report that ENDOG released from mitochondria promotes autophagy during starvation, which this study finds to be evolutionally conserved across species by performing experiments in human cell lines, mice, Drosophila and C. elegans. Under starvation, Glycogen synthase kinase 3 beta-mediated phosphorylation of ENDOG at Thr-128 and Ser-288 enhances its interaction with 14-3-3γ, which leads to the release of Tuberin (TSC2) and Phosphatidylinositol 3-kinase catalytic subunit type 3 (Vps34) from 14-3-3γ, followed by mTOR pathway suppression and autophagy initiation. Alternatively, ENDOG activates DNA damage response and triggers autophagy through its endonuclease activity. These results demonstrate that ENDOG is a crucial regulator of autophagy, manifested by phosphorylation-mediated interaction with 14-3-3γ, and its endonuclease activity-mediated DNA damage response.
Choi, H. J., Lee, J. Y., Cha, S. J., Han, Y. J., Yoon, J. H., Kim, H. J. and Kim, K. (2021). FUS-induced neurotoxicity is prevented by inhibiting GSK-3 model in a Drosophila model of amyotrophic lateral sclerosis. Hum Mol Genet. PubMed ID: 34605896
Summary:
Amyotrophic lateral sclerosis (ALS)-linked mutations in fused in sarcoma (FUS) lead to the formation of cytoplasmic aggregates in neurons. They are believed play a critical role in the pathogenesis of FUS-associated ALS. Therefore, the clearance and degradation of cytoplasmic FUS aggregates in neurons may be considered a therapeutic strategy for ALS. This study reports GSK-3β as a potential modulator of FUS-induced toxicity. RNAi-mediated knockdown of Drosophila ortholog Shaggy in FUS-expressing flies suppresses defective phenotypes, including retinal degeneration, motor defects, motor neuron degeneration, and mitochondrial dysfunction. Furthermore, it was found that cytoplasmic FUS aggregates were significantly reduced by Shaggy knockdown. In addition, studies found that the levels of FUS proteins were significantly reduced by co-overexpression of Slimb, a F-box protein, in FUS-expressing flies, indicating that Slimb is critical for the suppressive effect of Shaggy/GSK-3β inhibition on FUS-induced toxicity in Drosophila. These findings revealed a novel mechanism of neuronal protective effect through SCFSlimb-mediated FUS degradation via GSK-3β inhibition, and provided in vivo evidence of the potential for modulating FUS-induced ALS progression using GSK-3β inhibitors (Choi, 2021).
Choi, H. J., Lee, J. Y., Cha, S. J., Han, Y. J., Yoon, J. H., Kim, H. J. and Kim, K. (2021). FUS-induced neurotoxicity is prevented by inhibiting GSK-3beta in a Drosophila model of amyotrophic lateral sclerosis. Hum Mol Genet. PubMed ID: 34605896.
Summary:
Amyotrophic lateral sclerosis (ALS)-linked mutations in fused in sarcoma (FUS; see Drosophila Cabeza) lead to the formation of cytoplasmic aggregates in neurons. They are believed play a critical role in the pathogenesis of FUS-associated ALS. Therefore, the clearance and degradation of cytoplasmic FUS aggregates in neurons may be considered a therapeutic strategy for ALS. However, the molecular pathogenic mechanisms behind FUS-associated ALS remain poorly understood. This paper reports GSK-3β as a potential modulator of FUS-induced toxicity. RNAi-mediated knockdown of Drosophila ortholog Shaggy in FUS-expressing flies suppresses defective phenotypes, including retinal degeneration, motor defects, motor neuron degeneration, and mitochondrial dysfunction. Furthermore, cytoplasmic FUS aggregates were significantly reduced by Shaggy knockdown. In addition, it was found that the levels of FUS proteins were significantly reduced by co-overexpression of Slimb, a F-box protein, in FUS-expressing flies, indicating that Slimb is critical for the suppressive effect of Shaggy/GSK-3β inhibition on FUS-induced toxicity in Drosophila. These findings revealed a novel mechanism of neuronal protective effect through SCFSlimb-mediated FUS degradation via GSK-3β inhibition, and provided in vivo evidence of the potential for modulating FUS-induced ALS progression using GSK-3β inhibitors.
Graca, F. A., Sheffield, N., Puppa, M., Finkelstein, D., Hunt, L. C. and Demontis, F. (2021). A large-scale transgenic RNAi screen identifies transcription factors that modulate myofiber size in Drosophila. PLoS Genet 17(11): e1009926. PubMed ID: 34780463
Summary:
Myofiber atrophy occurs with aging and in many diseases but the underlying mechanisms are incompletely understood. This study used >1,100 muscle-targeted RNAi interventions to comprehensively assess the function of 447 transcription factors in the developmental growth of body wall skeletal muscles in Drosophila. This screen identifies new regulators of myofiber atrophy and hypertrophy, including the transcription factor Deaf1. Deaf1 RNAi increases myofiber size whereas Deaf1 overexpression induces atrophy. Consistent with its annotation as a Gsk3 phosphorylation substrate, Deaf1 and Gsk3 induce largely overlapping transcriptional changes that are opposed by Deaf1 RNAi. The top category of Deaf1-regulated genes consists of glycolytic enzymes, which are suppressed by Deaf1 and Gsk3 but are upregulated by Deaf1 RNAi. Similar to Deaf1 and Gsk3 overexpression, RNAi for glycolytic enzymes reduces myofiber growth. Altogether, this study defines the repertoire of transcription factors that regulate developmental myofiber growth and the role of Gsk3/Deaf1/glycolysis in this process.
Banerjee, R., Chakraborty, P., Yu, M. C. and Gunawardena, S. (2021). A stop or go switch: glycogen synthase kinase 3β phosphorylation of the kinesin 1 motor domain at Ser314 halts motility without detaching from microtubules. Development 148(24). PubMed ID: 34940839
Summary:
It is more than 25 years since the discovery that kinesin 1 is phosphorylated by several protein kinases. However, fundamental questions still remain as to how specific protein kinase(s) contribute to particular motor functions under physiological conditions. Because, within an whole organism, kinase cascades display considerable crosstalk and play multiple roles in cell homeostasis, deciphering which kinase(s) is/are involved in a particular process has been challenging. Previous work found that GSK3β plays a role in motor function. This study reports that a particular site on kinesin 1 motor domain (KHC), S314, is phosphorylated by GSK3β in vivo. The GSK3β-phosphomimetic-KHCS314D stalled kinesin 1 motility without dissociating from microtubules, indicating that constitutive GSK3β phosphorylation of the motor domain acts as a STOP. In contrast, uncoordinated mitochondrial motility was observed in CRISPR/Cas9-GSK3β non-phosphorylatable-KHCS314A Drosophila larval axons, owing to decreased kinesin 1 attachment to microtubules and/or membranes, and reduced ATPase activity. Together, it is proposed that GSK3β phosphorylation fine-tunes kinesin 1 movement in vivo via differential phosphorylation, unraveling the complex in vivo regulatory mechanisms that exist during axonal motility of cargos attached to multiple kinesin 1 and dynein motors.
Fang, Y., Chen, B., Liu, Z., Gong, A. Y., Gunning, W. T., Ge, Y., Malhotra, D., Gohara, A. F., Dworkin, L. D. and Gong, R. (2022). Age-related GSK3beta overexpression drives podocyte senescence and glomerular aging. J Clin Invest 132(4). PubMed ID: 35166234
Summary:
As life expectancy continues to increase, clinicians are challenged by age-related renal impairment that involves podocyte senescence and glomerulosclerosis. There is now compelling evidence that lithium has a potent antiaging activity that ameliorates brain aging and increases longevity in Drosophila and Caenorhabditis elegans. As the major molecular target of lithium action and a multitasking protein kinase recently implicated in a variety of renal diseases, glycogen synthase kinase 3β (GSK3β) is overexpressed and hyperactive with age in glomerular podocytes, correlating with functional and histological signs of kidney aging. Moreover, podocyte-specific ablation of GSK3β substantially attenuated podocyte senescence and glomerular aging in mice. Mechanistically, key mediators of senescence signaling, such as p16INK4A and p53, contain high numbers of GSK3β consensus motifs, physically interact with GSK3β, and act as its putative substrates. In addition, therapeutic targeting of GSK3β by microdose lithium later in life reduced senescence signaling and delayed kidney aging in mice. Furthermore, in psychiatric patients, lithium carbonate therapy inhibited GSK3β activity and mitigated senescence signaling in urinary exfoliated podocytes and was associated with preservation of kidney function. Thus, GSK3β appears to play a key role in podocyte senescence by modulating senescence signaling and may be an actionable senostatic target to delay kidney aging.
Fang, Y., Chen, B., Liu, Z., Gong, A. Y., Gunning, W. T., Ge, Y., Malhotra, D., Gohara, A. F., Dworkin, L. D. and Gong, R. (2022). Age-related GSK3beta overexpression drives podocyte senescence and glomerular aging. J Clin Invest 132(4). PubMed ID: 35166234
Summary:
As life expectancy continues to increase, clinicians are challenged by age-related renal impairment that involves podocyte senescence and glomerulosclerosis. There is now compelling evidence that lithium has a potent antiaging activity that ameliorates brain aging and increases longevity in Drosophila and Caenorhabditis elegans. As the major molecular target of lithium action and a multitasking protein kinase recently implicated in a variety of renal diseases, glycogen synthase kinase 3β (GSK3β) is overexpressed and hyperactive with age in glomerular podocytes, correlating with functional and histological signs of kidney aging. Moreover, podocyte-specific ablation of GSK3β substantially attenuated podocyte senescence and glomerular aging in mice. Mechanistically, key mediators of senescence signaling, such as p16INK4A and p53, contain high numbers of GSK3β consensus motifs, physically interact with GSK3β, and act as its putative substrates. In addition, therapeutic targeting of GSK3β by microdose lithium later in life reduced senescence signaling and delayed kidney aging in mice. Furthermore, in psychiatric patients, lithium carbonate therapy inhibited GSK3β activity and mitigated senescence signaling in urinary exfoliated podocytes and was associated with preservation of kidney function. Thus, GSK3β appears to play a key role in podocyte senescence by modulating senescence signaling and may be an actionable senostatic target to delay kidney aging.
Hope, K. A., Berman, A. R., Peterson, R. T. and Chow, C. Y. (2022). An in vivo drug repurposing screen and transcriptional analyses reveals the serotonin pathway and GSK3 as major therapeutic targets for NGLY1 deficiency. PLoS Genet 18(6): e1010228. PubMed ID: 35653343
Summary:
NGLY1 deficiency, a rare disease with no effective treatment, is caused by autosomal recessive, loss-of-function mutations in the N-glycanase 1 (NGLY1) gene and is characterized by global developmental delay, hypotonia, alacrima, and seizures. This study used a Drosophila model of NGLY1 deficiency to conduct an in vivo, unbiased, small molecule, repurposing screen of FDA-approved drugs to identify therapeutic compounds. Seventeen molecules partially rescued lethality in a patient-specific NGLY1 deficiency model, including multiple serotonin and dopamine modulators. Exclusive dNGLY1 expression in serotonin and dopamine neurons, in an otherwise dNGLY1 deficient fly, was sufficient to partially rescue lethality. Further, genetic modifier and transcriptomic data supports the importance of serotonin signaling in NGLY1 deficiency. Connectivity Map analysis identified glycogen synthase kinase 3 (GSK3) inhibition as a potential therapeutic mechanism for NGLY1 deficiency, which we experimentally validated with TWS119, lithium, and GSK3 knockdown. Strikingly, GSK3 inhibitors and a serotonin modulator rescued size defects in dNGLY1 deficient larvae upon proteasome inhibition, suggesting that these compounds act through NRF1, a transcription factor that is regulated by NGLY1 and regulates proteasome expression. This study reveals the importance of the serotonin pathway in NGLY1 deficiency, and serotonin modulators or GSK3 inhibitors may be effective therapeutics for this rare disease.
Dzaki, N., Bu, S., Lau, S. S. Y., Yong, W. L. and Yu, F. (2022). Drosophila GSK3β promotes microtubule disassembly and dendrite pruning in sensory neurons. Development 149(22). PubMed ID: 36264221
Summary:
The evolutionarily conserved Glycogen Synthase Kinase 3β (GSK3β), a negative regulator of microtubules, is crucial for neuronal polarization, growth and migration during animal development. However, it remains unknown whether GSK3β regulates neuronal pruning, which is a regressive process. This study reports that the Drosophila GSK3β homologue Shaggy (Sgg) is cell-autonomously required for dendrite pruning of ddaC sensory neurons during metamorphosis. Sgg is necessary and sufficient to promote microtubule depolymerization, turnover and disassembly in the dendrites. Although Sgg is not required for the minus-end-out microtubule orientation in dendrites, hyperactivated Sgg can disturb the dendritic microtubule orientation. Moreover, pharmacological and genetic data suggest that Sgg is required to promote dendrite pruning at least partly via microtubule disassembly. This study showed that Sgg and Par-1 kinases act synergistically to promote microtubule disassembly and dendrite pruning. Thus, Sgg and Par-1 might converge on and phosphorylate a common downstream microtubule-associated protein(s) to disassemble microtubules and thereby facilitate dendrite pruning.
Ataellahi, F., Masoudi, R. and Haddadi, M. (2022). Differential dysregulation of CREB and synaptic genes in transgenic Drosophila melanogaster expressing shaggy (GSK3), Tau(WT), or Amyloid-beta. Mol Biol Rep. PubMed ID: 36399243
Summary:
Tau, Amyloid-beta (Aβ42), and Glycogen synthase kinase 3 (GSK3) contribute to synaptic dysfunction observed in Alzheimer's disease (AD), the most common form of dementia. In the current study, the effect of pan-neuronal expression of TauWT, Aβ42, or shaggy (orthologue of GSK3) in Drosophila melanogaster was assessed on the locomotor function, ethanol sensitivity, synaptic genes and CREB expression. The effect of TauWT and Aβ42 on the expression of shaggy was also determined. Gene expression analysis was performed using quantitative real-time RT-PCR method. While syt1, SNAP25 and CREB (upstream transcription factor of syt1 and SNAP25) were upregulated in flies expressing Tau(WT) or Aβ42, a prominent decline was observed in those genes in shaggy expressing flies. Although all transgenic flies showed climbing disability and higher sensitivity to ethanol, abnormality in these features was significantly more prominent in transgenic flies expressing shaggy compared to TauWT or Aβ42. Despite a significant upregulation of shaggy transcription in TauWT expressing flies, Aβ42 transgenic flies witnessed no significant changes. TauWT, A&beta42, and shaggy may affect synaptic plasticity through dysregulation of synaptic genes and CREB, independently. However shaggy has more detrimental effect on synaptic genes expression, locomotor ability and sensitivity to ethanol. It is important when it comes to drug discovery. It appears that CREB is a direct effector of changes in synaptic genes expression as they showed similar pattern of alteration and it is likely to be a part of compensatory mechanisms independent of the GSK3/CREB pathway in TauWT or Aβ(42) expressing flies.
Wu, H., Zhu, N., Liu, J., Ma, J. and Jiao, R. (2022). Shaggy regulates tissue growth through Hippo pathway in Drosophila. Sci China Life Sci. PubMed ID: 36057002
Summary:
The evolutionarily conserved Hippo pathway coordinates cell proliferation, differentiation and apoptosis to regulate organ growth and tumorigenesis. Hippo signaling activity is tightly controlled by various upstream signals including growth factors and cell polarity, but the full extent to which the pathway is regulated during development remains to be resolved. This study reports the identification of Shaggy, the homolog of mammalian Gsk3β, as a novel regulator of the Hippo pathway in Drosophila. These results show that Shaggy promotes the expression of Hippo target genes in a manner that is dependent on its kinase activity. Loss of Shaggy leads to Yorkie inhibition and downregulation of Hippo pathway target genes. Mechanistically, Shaggy acts upstream of the Hippo pathway and negatively regulates the abundance of the FERM domain containing adaptor protein Expanded. These results reveal that Shaggy is functionally required for Crumbs/Slmb-mediated downregulation of Expanded in vivo, providing a potential molecular link between cellular architecture and the Hippo signaling pathway.

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 TORTED, 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 RhebAV4 . 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+, dmP0 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 Rheb7A1 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 RhebAV4 to induce cell death during the development of the eye imaginal disc. Rheb-induced cell death was rescued in a dmP0 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 APPswe 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).


shaggy: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 2 January 2023 

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